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Biophysics IIBiophysics II
ByByAProf Xiang Yang LiuAProf Xiang Yang LiuBiophysics LabBiophysics LabDepartment of Physics Department of Physics NUSNUS
Announcement
Term Test ndash Mar 2 2007 Fri 1048700 Venue S13-0507 1048700 Time 830-930am (1 hr) Closed book One A4 ldquocheat sheetrdquo is allowed
Outline
ELECTROPHORESIS Discussion you are supposed to prepare
your questions to be discussed in the lecture
Example
Example
Example
If the temperature of the dissolution of sugar crystals in water is not very far from the melting temperature (Tm) the solubility (xi
mole fraction) of sugar can be described in terms of the dissolution enthalpy (Hm) Treat the dissolution equilibrium as a special chemical equilibrium Derive the relationship between xi and Hm
ELECTROPHORESIS
Most of the remarkable advances in molecular biology over the past few decades would have been impossible without electrophoretic methods
ELECTROPHORESIS
The great majority of the polymers of biological interest are electrically charged
Polyelectrolytes are somewhat arbitrarily classified as ldquostrongrdquo or ldquoweakrdquo depending on the ionization constants of the acidic or basic groups
They may be strong polyacids such as the nucleic acids weak polybases such as poly-l-lysine or polyampholytes such as the proteins
ELECTROPHORESIS
electric charge differences can be used to separate and analyze mixtures of biopolymers electrophoretic methods are used in every area of biochemistry and molecular charged most important physical technique available to scientists working in these fields
Electrophoresis General Principles Electrophoresis The transport of particles by
an electrical field The charged molecule is not alone but in the
presence of many other charged particles and these will both influence the local field and interact with the macromolecule making analysis difficult
Diffusion through a solvent
The Diffusion coefficient and the fraction coefficient Diffusion coefficient introduced Albert Einstein
D = kTf
f friction coefficient-measures the resistance encountered by the molecule in moving through the solvent
Stokesrsquo Law for a sphere of radius a
fo = 6a
the viscosity of the solvent
Do = kT6a
Electrophoresis General Principles An idealized simplified situation an isolated charged
particle in a nonconducting medium
The force experienced by a particle in an electrical field is given by Coulombrsquos law
F = ZeE (E-electric field potential per unit length) The viscous resistance of the medium to the motion -
fv (f the frictional factor) The viscous resistance of the medium just balances
the driving force fv = F = ZeE
Electrophoresis General Principles
Electrophoretic mobility U (the ratio of velocity to the strength of the driving field)
U = vE = Zef If the particle happens to be spherical Stokesrsquos law
applies U = Ze6a
The zonal techniques In these methods a thin layer or zone of the macromolecule solution is electrophoresed through some kind of matrix
The matrix provides stability against convection In addition in many cases the matrix acts as a molecular sieve to aid in the separation of molecules on the basis of size
Electrophoresis General Principles The kind of supporting matrix used depends on the type of
molecules to be separated and on the desired basis for separation charge molecular weight or both
Almost all electrophoresis of biological macromolecules is at
present carried out on either polyacrylamide or agarose gels
Electrophoresis
Each macromolecular solution is applied in a thin layer in one well
If several components of different mobility are present they will separate during electrophoresis just as the zones of molecules of different sedimentation rate separate in zonal centrifugation
Usually a dye of high mobility is added its migration serves to mark the progress of the experiment
Electrophoresis
The dye also serves as a convenient measure of mobility the relative mobility Uri of each component i is defined by
Ud is the dye mobility and di and dd are the distances that component i and dye respectively have moved by the conclusion of the experiment
Ferguson plots
A very simple relationship between relative mobility and gel concentration
logUri = logUrio - kiC
where C is the gel concentration and is the relative mobility of component i when C = 0 that is the relative mobility in free electrophoresis The constant ki large molecules will have large values of k whereas very small molecules will have small values and hence will behave almost the same way in a gel as they do in free electrophoresis
Electrophoresis
Ferguson plots for a number of commonly encountered solutions
Example
Analysis of multisubunit structures by SDS gel electrophoresis a very widely used technique for the estimation of the
molecular weights of polypeptide chains the protein to be studied is first heated in a dilute
solution of a detergent such as sodium dodecyl sulfate (SDS) This breaks down all native quaternary tertiary and secondary structures in the protein Usually a reducing agent such as -mercaptoethanol is also
added to reduce any disulfide bonds The protein is then electrophoresed in the presence of SDS The separation proceeds on the basis of polypeptide-chain
weight and is nearly independent of the charge on the polypeptide
Analysis of multisubunit structures by SDS gel electrophoresisGeneral principles First SDS at a given solution concentration binds to
many different proteins at a constant weight-weight ratio there is a defined number of bound SDS charges per amino acid residue therefore the charge contributed by SDS is proportional to protein molecular weight
the complexes between SDS and proteins are extended structures molecules with both charge and friction proportional to molecular weight As in that case this predicts that the free mobility will be essentially independent of molecular weight and that separation will be by the effect of sieving
Analysis of multisubunit structures by SDS gel electrophoresis As in the case of DNA
there exists for each particular gel type and concentration an approximately linear relationship between the logarithm of the protein molecular weight and mobility
logUri = logUrio - kiC
DNA samples (open squares) are restriction fragments from a bacterial plasmid Proteins are polymers of the globular domain of histone H5 cross- linked with either glutaraldehyde (squares) or dithiobis (succinimidyl propionate) (circles)
References
Section 54 in Principles of Physical Biochemistry
Announcement
Term Test ndash Mar 2 2007 Fri 1048700 Venue S13-0507 1048700 Time 830-930am (1 hr) Closed book One A4 ldquocheat sheetrdquo is allowed
Outline
ELECTROPHORESIS Discussion you are supposed to prepare
your questions to be discussed in the lecture
Example
Example
Example
If the temperature of the dissolution of sugar crystals in water is not very far from the melting temperature (Tm) the solubility (xi
mole fraction) of sugar can be described in terms of the dissolution enthalpy (Hm) Treat the dissolution equilibrium as a special chemical equilibrium Derive the relationship between xi and Hm
ELECTROPHORESIS
Most of the remarkable advances in molecular biology over the past few decades would have been impossible without electrophoretic methods
ELECTROPHORESIS
The great majority of the polymers of biological interest are electrically charged
Polyelectrolytes are somewhat arbitrarily classified as ldquostrongrdquo or ldquoweakrdquo depending on the ionization constants of the acidic or basic groups
They may be strong polyacids such as the nucleic acids weak polybases such as poly-l-lysine or polyampholytes such as the proteins
ELECTROPHORESIS
electric charge differences can be used to separate and analyze mixtures of biopolymers electrophoretic methods are used in every area of biochemistry and molecular charged most important physical technique available to scientists working in these fields
Electrophoresis General Principles Electrophoresis The transport of particles by
an electrical field The charged molecule is not alone but in the
presence of many other charged particles and these will both influence the local field and interact with the macromolecule making analysis difficult
Diffusion through a solvent
The Diffusion coefficient and the fraction coefficient Diffusion coefficient introduced Albert Einstein
D = kTf
f friction coefficient-measures the resistance encountered by the molecule in moving through the solvent
Stokesrsquo Law for a sphere of radius a
fo = 6a
the viscosity of the solvent
Do = kT6a
Electrophoresis General Principles An idealized simplified situation an isolated charged
particle in a nonconducting medium
The force experienced by a particle in an electrical field is given by Coulombrsquos law
F = ZeE (E-electric field potential per unit length) The viscous resistance of the medium to the motion -
fv (f the frictional factor) The viscous resistance of the medium just balances
the driving force fv = F = ZeE
Electrophoresis General Principles
Electrophoretic mobility U (the ratio of velocity to the strength of the driving field)
U = vE = Zef If the particle happens to be spherical Stokesrsquos law
applies U = Ze6a
The zonal techniques In these methods a thin layer or zone of the macromolecule solution is electrophoresed through some kind of matrix
The matrix provides stability against convection In addition in many cases the matrix acts as a molecular sieve to aid in the separation of molecules on the basis of size
Electrophoresis General Principles The kind of supporting matrix used depends on the type of
molecules to be separated and on the desired basis for separation charge molecular weight or both
Almost all electrophoresis of biological macromolecules is at
present carried out on either polyacrylamide or agarose gels
Electrophoresis
Each macromolecular solution is applied in a thin layer in one well
If several components of different mobility are present they will separate during electrophoresis just as the zones of molecules of different sedimentation rate separate in zonal centrifugation
Usually a dye of high mobility is added its migration serves to mark the progress of the experiment
Electrophoresis
The dye also serves as a convenient measure of mobility the relative mobility Uri of each component i is defined by
Ud is the dye mobility and di and dd are the distances that component i and dye respectively have moved by the conclusion of the experiment
Ferguson plots
A very simple relationship between relative mobility and gel concentration
logUri = logUrio - kiC
where C is the gel concentration and is the relative mobility of component i when C = 0 that is the relative mobility in free electrophoresis The constant ki large molecules will have large values of k whereas very small molecules will have small values and hence will behave almost the same way in a gel as they do in free electrophoresis
Electrophoresis
Ferguson plots for a number of commonly encountered solutions
Example
Analysis of multisubunit structures by SDS gel electrophoresis a very widely used technique for the estimation of the
molecular weights of polypeptide chains the protein to be studied is first heated in a dilute
solution of a detergent such as sodium dodecyl sulfate (SDS) This breaks down all native quaternary tertiary and secondary structures in the protein Usually a reducing agent such as -mercaptoethanol is also
added to reduce any disulfide bonds The protein is then electrophoresed in the presence of SDS The separation proceeds on the basis of polypeptide-chain
weight and is nearly independent of the charge on the polypeptide
Analysis of multisubunit structures by SDS gel electrophoresisGeneral principles First SDS at a given solution concentration binds to
many different proteins at a constant weight-weight ratio there is a defined number of bound SDS charges per amino acid residue therefore the charge contributed by SDS is proportional to protein molecular weight
the complexes between SDS and proteins are extended structures molecules with both charge and friction proportional to molecular weight As in that case this predicts that the free mobility will be essentially independent of molecular weight and that separation will be by the effect of sieving
Analysis of multisubunit structures by SDS gel electrophoresis As in the case of DNA
there exists for each particular gel type and concentration an approximately linear relationship between the logarithm of the protein molecular weight and mobility
logUri = logUrio - kiC
DNA samples (open squares) are restriction fragments from a bacterial plasmid Proteins are polymers of the globular domain of histone H5 cross- linked with either glutaraldehyde (squares) or dithiobis (succinimidyl propionate) (circles)
References
Section 54 in Principles of Physical Biochemistry
Outline
ELECTROPHORESIS Discussion you are supposed to prepare
your questions to be discussed in the lecture
Example
Example
Example
If the temperature of the dissolution of sugar crystals in water is not very far from the melting temperature (Tm) the solubility (xi
mole fraction) of sugar can be described in terms of the dissolution enthalpy (Hm) Treat the dissolution equilibrium as a special chemical equilibrium Derive the relationship between xi and Hm
ELECTROPHORESIS
Most of the remarkable advances in molecular biology over the past few decades would have been impossible without electrophoretic methods
ELECTROPHORESIS
The great majority of the polymers of biological interest are electrically charged
Polyelectrolytes are somewhat arbitrarily classified as ldquostrongrdquo or ldquoweakrdquo depending on the ionization constants of the acidic or basic groups
They may be strong polyacids such as the nucleic acids weak polybases such as poly-l-lysine or polyampholytes such as the proteins
ELECTROPHORESIS
electric charge differences can be used to separate and analyze mixtures of biopolymers electrophoretic methods are used in every area of biochemistry and molecular charged most important physical technique available to scientists working in these fields
Electrophoresis General Principles Electrophoresis The transport of particles by
an electrical field The charged molecule is not alone but in the
presence of many other charged particles and these will both influence the local field and interact with the macromolecule making analysis difficult
Diffusion through a solvent
The Diffusion coefficient and the fraction coefficient Diffusion coefficient introduced Albert Einstein
D = kTf
f friction coefficient-measures the resistance encountered by the molecule in moving through the solvent
Stokesrsquo Law for a sphere of radius a
fo = 6a
the viscosity of the solvent
Do = kT6a
Electrophoresis General Principles An idealized simplified situation an isolated charged
particle in a nonconducting medium
The force experienced by a particle in an electrical field is given by Coulombrsquos law
F = ZeE (E-electric field potential per unit length) The viscous resistance of the medium to the motion -
fv (f the frictional factor) The viscous resistance of the medium just balances
the driving force fv = F = ZeE
Electrophoresis General Principles
Electrophoretic mobility U (the ratio of velocity to the strength of the driving field)
U = vE = Zef If the particle happens to be spherical Stokesrsquos law
applies U = Ze6a
The zonal techniques In these methods a thin layer or zone of the macromolecule solution is electrophoresed through some kind of matrix
The matrix provides stability against convection In addition in many cases the matrix acts as a molecular sieve to aid in the separation of molecules on the basis of size
Electrophoresis General Principles The kind of supporting matrix used depends on the type of
molecules to be separated and on the desired basis for separation charge molecular weight or both
Almost all electrophoresis of biological macromolecules is at
present carried out on either polyacrylamide or agarose gels
Electrophoresis
Each macromolecular solution is applied in a thin layer in one well
If several components of different mobility are present they will separate during electrophoresis just as the zones of molecules of different sedimentation rate separate in zonal centrifugation
Usually a dye of high mobility is added its migration serves to mark the progress of the experiment
Electrophoresis
The dye also serves as a convenient measure of mobility the relative mobility Uri of each component i is defined by
Ud is the dye mobility and di and dd are the distances that component i and dye respectively have moved by the conclusion of the experiment
Ferguson plots
A very simple relationship between relative mobility and gel concentration
logUri = logUrio - kiC
where C is the gel concentration and is the relative mobility of component i when C = 0 that is the relative mobility in free electrophoresis The constant ki large molecules will have large values of k whereas very small molecules will have small values and hence will behave almost the same way in a gel as they do in free electrophoresis
Electrophoresis
Ferguson plots for a number of commonly encountered solutions
Example
Analysis of multisubunit structures by SDS gel electrophoresis a very widely used technique for the estimation of the
molecular weights of polypeptide chains the protein to be studied is first heated in a dilute
solution of a detergent such as sodium dodecyl sulfate (SDS) This breaks down all native quaternary tertiary and secondary structures in the protein Usually a reducing agent such as -mercaptoethanol is also
added to reduce any disulfide bonds The protein is then electrophoresed in the presence of SDS The separation proceeds on the basis of polypeptide-chain
weight and is nearly independent of the charge on the polypeptide
Analysis of multisubunit structures by SDS gel electrophoresisGeneral principles First SDS at a given solution concentration binds to
many different proteins at a constant weight-weight ratio there is a defined number of bound SDS charges per amino acid residue therefore the charge contributed by SDS is proportional to protein molecular weight
the complexes between SDS and proteins are extended structures molecules with both charge and friction proportional to molecular weight As in that case this predicts that the free mobility will be essentially independent of molecular weight and that separation will be by the effect of sieving
Analysis of multisubunit structures by SDS gel electrophoresis As in the case of DNA
there exists for each particular gel type and concentration an approximately linear relationship between the logarithm of the protein molecular weight and mobility
logUri = logUrio - kiC
DNA samples (open squares) are restriction fragments from a bacterial plasmid Proteins are polymers of the globular domain of histone H5 cross- linked with either glutaraldehyde (squares) or dithiobis (succinimidyl propionate) (circles)
References
Section 54 in Principles of Physical Biochemistry
Example
Example
Example
If the temperature of the dissolution of sugar crystals in water is not very far from the melting temperature (Tm) the solubility (xi
mole fraction) of sugar can be described in terms of the dissolution enthalpy (Hm) Treat the dissolution equilibrium as a special chemical equilibrium Derive the relationship between xi and Hm
ELECTROPHORESIS
Most of the remarkable advances in molecular biology over the past few decades would have been impossible without electrophoretic methods
ELECTROPHORESIS
The great majority of the polymers of biological interest are electrically charged
Polyelectrolytes are somewhat arbitrarily classified as ldquostrongrdquo or ldquoweakrdquo depending on the ionization constants of the acidic or basic groups
They may be strong polyacids such as the nucleic acids weak polybases such as poly-l-lysine or polyampholytes such as the proteins
ELECTROPHORESIS
electric charge differences can be used to separate and analyze mixtures of biopolymers electrophoretic methods are used in every area of biochemistry and molecular charged most important physical technique available to scientists working in these fields
Electrophoresis General Principles Electrophoresis The transport of particles by
an electrical field The charged molecule is not alone but in the
presence of many other charged particles and these will both influence the local field and interact with the macromolecule making analysis difficult
Diffusion through a solvent
The Diffusion coefficient and the fraction coefficient Diffusion coefficient introduced Albert Einstein
D = kTf
f friction coefficient-measures the resistance encountered by the molecule in moving through the solvent
Stokesrsquo Law for a sphere of radius a
fo = 6a
the viscosity of the solvent
Do = kT6a
Electrophoresis General Principles An idealized simplified situation an isolated charged
particle in a nonconducting medium
The force experienced by a particle in an electrical field is given by Coulombrsquos law
F = ZeE (E-electric field potential per unit length) The viscous resistance of the medium to the motion -
fv (f the frictional factor) The viscous resistance of the medium just balances
the driving force fv = F = ZeE
Electrophoresis General Principles
Electrophoretic mobility U (the ratio of velocity to the strength of the driving field)
U = vE = Zef If the particle happens to be spherical Stokesrsquos law
applies U = Ze6a
The zonal techniques In these methods a thin layer or zone of the macromolecule solution is electrophoresed through some kind of matrix
The matrix provides stability against convection In addition in many cases the matrix acts as a molecular sieve to aid in the separation of molecules on the basis of size
Electrophoresis General Principles The kind of supporting matrix used depends on the type of
molecules to be separated and on the desired basis for separation charge molecular weight or both
Almost all electrophoresis of biological macromolecules is at
present carried out on either polyacrylamide or agarose gels
Electrophoresis
Each macromolecular solution is applied in a thin layer in one well
If several components of different mobility are present they will separate during electrophoresis just as the zones of molecules of different sedimentation rate separate in zonal centrifugation
Usually a dye of high mobility is added its migration serves to mark the progress of the experiment
Electrophoresis
The dye also serves as a convenient measure of mobility the relative mobility Uri of each component i is defined by
Ud is the dye mobility and di and dd are the distances that component i and dye respectively have moved by the conclusion of the experiment
Ferguson plots
A very simple relationship between relative mobility and gel concentration
logUri = logUrio - kiC
where C is the gel concentration and is the relative mobility of component i when C = 0 that is the relative mobility in free electrophoresis The constant ki large molecules will have large values of k whereas very small molecules will have small values and hence will behave almost the same way in a gel as they do in free electrophoresis
Electrophoresis
Ferguson plots for a number of commonly encountered solutions
Example
Analysis of multisubunit structures by SDS gel electrophoresis a very widely used technique for the estimation of the
molecular weights of polypeptide chains the protein to be studied is first heated in a dilute
solution of a detergent such as sodium dodecyl sulfate (SDS) This breaks down all native quaternary tertiary and secondary structures in the protein Usually a reducing agent such as -mercaptoethanol is also
added to reduce any disulfide bonds The protein is then electrophoresed in the presence of SDS The separation proceeds on the basis of polypeptide-chain
weight and is nearly independent of the charge on the polypeptide
Analysis of multisubunit structures by SDS gel electrophoresisGeneral principles First SDS at a given solution concentration binds to
many different proteins at a constant weight-weight ratio there is a defined number of bound SDS charges per amino acid residue therefore the charge contributed by SDS is proportional to protein molecular weight
the complexes between SDS and proteins are extended structures molecules with both charge and friction proportional to molecular weight As in that case this predicts that the free mobility will be essentially independent of molecular weight and that separation will be by the effect of sieving
Analysis of multisubunit structures by SDS gel electrophoresis As in the case of DNA
there exists for each particular gel type and concentration an approximately linear relationship between the logarithm of the protein molecular weight and mobility
logUri = logUrio - kiC
DNA samples (open squares) are restriction fragments from a bacterial plasmid Proteins are polymers of the globular domain of histone H5 cross- linked with either glutaraldehyde (squares) or dithiobis (succinimidyl propionate) (circles)
References
Section 54 in Principles of Physical Biochemistry
Example
Example
If the temperature of the dissolution of sugar crystals in water is not very far from the melting temperature (Tm) the solubility (xi
mole fraction) of sugar can be described in terms of the dissolution enthalpy (Hm) Treat the dissolution equilibrium as a special chemical equilibrium Derive the relationship between xi and Hm
ELECTROPHORESIS
Most of the remarkable advances in molecular biology over the past few decades would have been impossible without electrophoretic methods
ELECTROPHORESIS
The great majority of the polymers of biological interest are electrically charged
Polyelectrolytes are somewhat arbitrarily classified as ldquostrongrdquo or ldquoweakrdquo depending on the ionization constants of the acidic or basic groups
They may be strong polyacids such as the nucleic acids weak polybases such as poly-l-lysine or polyampholytes such as the proteins
ELECTROPHORESIS
electric charge differences can be used to separate and analyze mixtures of biopolymers electrophoretic methods are used in every area of biochemistry and molecular charged most important physical technique available to scientists working in these fields
Electrophoresis General Principles Electrophoresis The transport of particles by
an electrical field The charged molecule is not alone but in the
presence of many other charged particles and these will both influence the local field and interact with the macromolecule making analysis difficult
Diffusion through a solvent
The Diffusion coefficient and the fraction coefficient Diffusion coefficient introduced Albert Einstein
D = kTf
f friction coefficient-measures the resistance encountered by the molecule in moving through the solvent
Stokesrsquo Law for a sphere of radius a
fo = 6a
the viscosity of the solvent
Do = kT6a
Electrophoresis General Principles An idealized simplified situation an isolated charged
particle in a nonconducting medium
The force experienced by a particle in an electrical field is given by Coulombrsquos law
F = ZeE (E-electric field potential per unit length) The viscous resistance of the medium to the motion -
fv (f the frictional factor) The viscous resistance of the medium just balances
the driving force fv = F = ZeE
Electrophoresis General Principles
Electrophoretic mobility U (the ratio of velocity to the strength of the driving field)
U = vE = Zef If the particle happens to be spherical Stokesrsquos law
applies U = Ze6a
The zonal techniques In these methods a thin layer or zone of the macromolecule solution is electrophoresed through some kind of matrix
The matrix provides stability against convection In addition in many cases the matrix acts as a molecular sieve to aid in the separation of molecules on the basis of size
Electrophoresis General Principles The kind of supporting matrix used depends on the type of
molecules to be separated and on the desired basis for separation charge molecular weight or both
Almost all electrophoresis of biological macromolecules is at
present carried out on either polyacrylamide or agarose gels
Electrophoresis
Each macromolecular solution is applied in a thin layer in one well
If several components of different mobility are present they will separate during electrophoresis just as the zones of molecules of different sedimentation rate separate in zonal centrifugation
Usually a dye of high mobility is added its migration serves to mark the progress of the experiment
Electrophoresis
The dye also serves as a convenient measure of mobility the relative mobility Uri of each component i is defined by
Ud is the dye mobility and di and dd are the distances that component i and dye respectively have moved by the conclusion of the experiment
Ferguson plots
A very simple relationship between relative mobility and gel concentration
logUri = logUrio - kiC
where C is the gel concentration and is the relative mobility of component i when C = 0 that is the relative mobility in free electrophoresis The constant ki large molecules will have large values of k whereas very small molecules will have small values and hence will behave almost the same way in a gel as they do in free electrophoresis
Electrophoresis
Ferguson plots for a number of commonly encountered solutions
Example
Analysis of multisubunit structures by SDS gel electrophoresis a very widely used technique for the estimation of the
molecular weights of polypeptide chains the protein to be studied is first heated in a dilute
solution of a detergent such as sodium dodecyl sulfate (SDS) This breaks down all native quaternary tertiary and secondary structures in the protein Usually a reducing agent such as -mercaptoethanol is also
added to reduce any disulfide bonds The protein is then electrophoresed in the presence of SDS The separation proceeds on the basis of polypeptide-chain
weight and is nearly independent of the charge on the polypeptide
Analysis of multisubunit structures by SDS gel electrophoresisGeneral principles First SDS at a given solution concentration binds to
many different proteins at a constant weight-weight ratio there is a defined number of bound SDS charges per amino acid residue therefore the charge contributed by SDS is proportional to protein molecular weight
the complexes between SDS and proteins are extended structures molecules with both charge and friction proportional to molecular weight As in that case this predicts that the free mobility will be essentially independent of molecular weight and that separation will be by the effect of sieving
Analysis of multisubunit structures by SDS gel electrophoresis As in the case of DNA
there exists for each particular gel type and concentration an approximately linear relationship between the logarithm of the protein molecular weight and mobility
logUri = logUrio - kiC
DNA samples (open squares) are restriction fragments from a bacterial plasmid Proteins are polymers of the globular domain of histone H5 cross- linked with either glutaraldehyde (squares) or dithiobis (succinimidyl propionate) (circles)
References
Section 54 in Principles of Physical Biochemistry
Example
If the temperature of the dissolution of sugar crystals in water is not very far from the melting temperature (Tm) the solubility (xi
mole fraction) of sugar can be described in terms of the dissolution enthalpy (Hm) Treat the dissolution equilibrium as a special chemical equilibrium Derive the relationship between xi and Hm
ELECTROPHORESIS
Most of the remarkable advances in molecular biology over the past few decades would have been impossible without electrophoretic methods
ELECTROPHORESIS
The great majority of the polymers of biological interest are electrically charged
Polyelectrolytes are somewhat arbitrarily classified as ldquostrongrdquo or ldquoweakrdquo depending on the ionization constants of the acidic or basic groups
They may be strong polyacids such as the nucleic acids weak polybases such as poly-l-lysine or polyampholytes such as the proteins
ELECTROPHORESIS
electric charge differences can be used to separate and analyze mixtures of biopolymers electrophoretic methods are used in every area of biochemistry and molecular charged most important physical technique available to scientists working in these fields
Electrophoresis General Principles Electrophoresis The transport of particles by
an electrical field The charged molecule is not alone but in the
presence of many other charged particles and these will both influence the local field and interact with the macromolecule making analysis difficult
Diffusion through a solvent
The Diffusion coefficient and the fraction coefficient Diffusion coefficient introduced Albert Einstein
D = kTf
f friction coefficient-measures the resistance encountered by the molecule in moving through the solvent
Stokesrsquo Law for a sphere of radius a
fo = 6a
the viscosity of the solvent
Do = kT6a
Electrophoresis General Principles An idealized simplified situation an isolated charged
particle in a nonconducting medium
The force experienced by a particle in an electrical field is given by Coulombrsquos law
F = ZeE (E-electric field potential per unit length) The viscous resistance of the medium to the motion -
fv (f the frictional factor) The viscous resistance of the medium just balances
the driving force fv = F = ZeE
Electrophoresis General Principles
Electrophoretic mobility U (the ratio of velocity to the strength of the driving field)
U = vE = Zef If the particle happens to be spherical Stokesrsquos law
applies U = Ze6a
The zonal techniques In these methods a thin layer or zone of the macromolecule solution is electrophoresed through some kind of matrix
The matrix provides stability against convection In addition in many cases the matrix acts as a molecular sieve to aid in the separation of molecules on the basis of size
Electrophoresis General Principles The kind of supporting matrix used depends on the type of
molecules to be separated and on the desired basis for separation charge molecular weight or both
Almost all electrophoresis of biological macromolecules is at
present carried out on either polyacrylamide or agarose gels
Electrophoresis
Each macromolecular solution is applied in a thin layer in one well
If several components of different mobility are present they will separate during electrophoresis just as the zones of molecules of different sedimentation rate separate in zonal centrifugation
Usually a dye of high mobility is added its migration serves to mark the progress of the experiment
Electrophoresis
The dye also serves as a convenient measure of mobility the relative mobility Uri of each component i is defined by
Ud is the dye mobility and di and dd are the distances that component i and dye respectively have moved by the conclusion of the experiment
Ferguson plots
A very simple relationship between relative mobility and gel concentration
logUri = logUrio - kiC
where C is the gel concentration and is the relative mobility of component i when C = 0 that is the relative mobility in free electrophoresis The constant ki large molecules will have large values of k whereas very small molecules will have small values and hence will behave almost the same way in a gel as they do in free electrophoresis
Electrophoresis
Ferguson plots for a number of commonly encountered solutions
Example
Analysis of multisubunit structures by SDS gel electrophoresis a very widely used technique for the estimation of the
molecular weights of polypeptide chains the protein to be studied is first heated in a dilute
solution of a detergent such as sodium dodecyl sulfate (SDS) This breaks down all native quaternary tertiary and secondary structures in the protein Usually a reducing agent such as -mercaptoethanol is also
added to reduce any disulfide bonds The protein is then electrophoresed in the presence of SDS The separation proceeds on the basis of polypeptide-chain
weight and is nearly independent of the charge on the polypeptide
Analysis of multisubunit structures by SDS gel electrophoresisGeneral principles First SDS at a given solution concentration binds to
many different proteins at a constant weight-weight ratio there is a defined number of bound SDS charges per amino acid residue therefore the charge contributed by SDS is proportional to protein molecular weight
the complexes between SDS and proteins are extended structures molecules with both charge and friction proportional to molecular weight As in that case this predicts that the free mobility will be essentially independent of molecular weight and that separation will be by the effect of sieving
Analysis of multisubunit structures by SDS gel electrophoresis As in the case of DNA
there exists for each particular gel type and concentration an approximately linear relationship between the logarithm of the protein molecular weight and mobility
logUri = logUrio - kiC
DNA samples (open squares) are restriction fragments from a bacterial plasmid Proteins are polymers of the globular domain of histone H5 cross- linked with either glutaraldehyde (squares) or dithiobis (succinimidyl propionate) (circles)
References
Section 54 in Principles of Physical Biochemistry
ELECTROPHORESIS
Most of the remarkable advances in molecular biology over the past few decades would have been impossible without electrophoretic methods
ELECTROPHORESIS
The great majority of the polymers of biological interest are electrically charged
Polyelectrolytes are somewhat arbitrarily classified as ldquostrongrdquo or ldquoweakrdquo depending on the ionization constants of the acidic or basic groups
They may be strong polyacids such as the nucleic acids weak polybases such as poly-l-lysine or polyampholytes such as the proteins
ELECTROPHORESIS
electric charge differences can be used to separate and analyze mixtures of biopolymers electrophoretic methods are used in every area of biochemistry and molecular charged most important physical technique available to scientists working in these fields
Electrophoresis General Principles Electrophoresis The transport of particles by
an electrical field The charged molecule is not alone but in the
presence of many other charged particles and these will both influence the local field and interact with the macromolecule making analysis difficult
Diffusion through a solvent
The Diffusion coefficient and the fraction coefficient Diffusion coefficient introduced Albert Einstein
D = kTf
f friction coefficient-measures the resistance encountered by the molecule in moving through the solvent
Stokesrsquo Law for a sphere of radius a
fo = 6a
the viscosity of the solvent
Do = kT6a
Electrophoresis General Principles An idealized simplified situation an isolated charged
particle in a nonconducting medium
The force experienced by a particle in an electrical field is given by Coulombrsquos law
F = ZeE (E-electric field potential per unit length) The viscous resistance of the medium to the motion -
fv (f the frictional factor) The viscous resistance of the medium just balances
the driving force fv = F = ZeE
Electrophoresis General Principles
Electrophoretic mobility U (the ratio of velocity to the strength of the driving field)
U = vE = Zef If the particle happens to be spherical Stokesrsquos law
applies U = Ze6a
The zonal techniques In these methods a thin layer or zone of the macromolecule solution is electrophoresed through some kind of matrix
The matrix provides stability against convection In addition in many cases the matrix acts as a molecular sieve to aid in the separation of molecules on the basis of size
Electrophoresis General Principles The kind of supporting matrix used depends on the type of
molecules to be separated and on the desired basis for separation charge molecular weight or both
Almost all electrophoresis of biological macromolecules is at
present carried out on either polyacrylamide or agarose gels
Electrophoresis
Each macromolecular solution is applied in a thin layer in one well
If several components of different mobility are present they will separate during electrophoresis just as the zones of molecules of different sedimentation rate separate in zonal centrifugation
Usually a dye of high mobility is added its migration serves to mark the progress of the experiment
Electrophoresis
The dye also serves as a convenient measure of mobility the relative mobility Uri of each component i is defined by
Ud is the dye mobility and di and dd are the distances that component i and dye respectively have moved by the conclusion of the experiment
Ferguson plots
A very simple relationship between relative mobility and gel concentration
logUri = logUrio - kiC
where C is the gel concentration and is the relative mobility of component i when C = 0 that is the relative mobility in free electrophoresis The constant ki large molecules will have large values of k whereas very small molecules will have small values and hence will behave almost the same way in a gel as they do in free electrophoresis
Electrophoresis
Ferguson plots for a number of commonly encountered solutions
Example
Analysis of multisubunit structures by SDS gel electrophoresis a very widely used technique for the estimation of the
molecular weights of polypeptide chains the protein to be studied is first heated in a dilute
solution of a detergent such as sodium dodecyl sulfate (SDS) This breaks down all native quaternary tertiary and secondary structures in the protein Usually a reducing agent such as -mercaptoethanol is also
added to reduce any disulfide bonds The protein is then electrophoresed in the presence of SDS The separation proceeds on the basis of polypeptide-chain
weight and is nearly independent of the charge on the polypeptide
Analysis of multisubunit structures by SDS gel electrophoresisGeneral principles First SDS at a given solution concentration binds to
many different proteins at a constant weight-weight ratio there is a defined number of bound SDS charges per amino acid residue therefore the charge contributed by SDS is proportional to protein molecular weight
the complexes between SDS and proteins are extended structures molecules with both charge and friction proportional to molecular weight As in that case this predicts that the free mobility will be essentially independent of molecular weight and that separation will be by the effect of sieving
Analysis of multisubunit structures by SDS gel electrophoresis As in the case of DNA
there exists for each particular gel type and concentration an approximately linear relationship between the logarithm of the protein molecular weight and mobility
logUri = logUrio - kiC
DNA samples (open squares) are restriction fragments from a bacterial plasmid Proteins are polymers of the globular domain of histone H5 cross- linked with either glutaraldehyde (squares) or dithiobis (succinimidyl propionate) (circles)
References
Section 54 in Principles of Physical Biochemistry
ELECTROPHORESIS
The great majority of the polymers of biological interest are electrically charged
Polyelectrolytes are somewhat arbitrarily classified as ldquostrongrdquo or ldquoweakrdquo depending on the ionization constants of the acidic or basic groups
They may be strong polyacids such as the nucleic acids weak polybases such as poly-l-lysine or polyampholytes such as the proteins
ELECTROPHORESIS
electric charge differences can be used to separate and analyze mixtures of biopolymers electrophoretic methods are used in every area of biochemistry and molecular charged most important physical technique available to scientists working in these fields
Electrophoresis General Principles Electrophoresis The transport of particles by
an electrical field The charged molecule is not alone but in the
presence of many other charged particles and these will both influence the local field and interact with the macromolecule making analysis difficult
Diffusion through a solvent
The Diffusion coefficient and the fraction coefficient Diffusion coefficient introduced Albert Einstein
D = kTf
f friction coefficient-measures the resistance encountered by the molecule in moving through the solvent
Stokesrsquo Law for a sphere of radius a
fo = 6a
the viscosity of the solvent
Do = kT6a
Electrophoresis General Principles An idealized simplified situation an isolated charged
particle in a nonconducting medium
The force experienced by a particle in an electrical field is given by Coulombrsquos law
F = ZeE (E-electric field potential per unit length) The viscous resistance of the medium to the motion -
fv (f the frictional factor) The viscous resistance of the medium just balances
the driving force fv = F = ZeE
Electrophoresis General Principles
Electrophoretic mobility U (the ratio of velocity to the strength of the driving field)
U = vE = Zef If the particle happens to be spherical Stokesrsquos law
applies U = Ze6a
The zonal techniques In these methods a thin layer or zone of the macromolecule solution is electrophoresed through some kind of matrix
The matrix provides stability against convection In addition in many cases the matrix acts as a molecular sieve to aid in the separation of molecules on the basis of size
Electrophoresis General Principles The kind of supporting matrix used depends on the type of
molecules to be separated and on the desired basis for separation charge molecular weight or both
Almost all electrophoresis of biological macromolecules is at
present carried out on either polyacrylamide or agarose gels
Electrophoresis
Each macromolecular solution is applied in a thin layer in one well
If several components of different mobility are present they will separate during electrophoresis just as the zones of molecules of different sedimentation rate separate in zonal centrifugation
Usually a dye of high mobility is added its migration serves to mark the progress of the experiment
Electrophoresis
The dye also serves as a convenient measure of mobility the relative mobility Uri of each component i is defined by
Ud is the dye mobility and di and dd are the distances that component i and dye respectively have moved by the conclusion of the experiment
Ferguson plots
A very simple relationship between relative mobility and gel concentration
logUri = logUrio - kiC
where C is the gel concentration and is the relative mobility of component i when C = 0 that is the relative mobility in free electrophoresis The constant ki large molecules will have large values of k whereas very small molecules will have small values and hence will behave almost the same way in a gel as they do in free electrophoresis
Electrophoresis
Ferguson plots for a number of commonly encountered solutions
Example
Analysis of multisubunit structures by SDS gel electrophoresis a very widely used technique for the estimation of the
molecular weights of polypeptide chains the protein to be studied is first heated in a dilute
solution of a detergent such as sodium dodecyl sulfate (SDS) This breaks down all native quaternary tertiary and secondary structures in the protein Usually a reducing agent such as -mercaptoethanol is also
added to reduce any disulfide bonds The protein is then electrophoresed in the presence of SDS The separation proceeds on the basis of polypeptide-chain
weight and is nearly independent of the charge on the polypeptide
Analysis of multisubunit structures by SDS gel electrophoresisGeneral principles First SDS at a given solution concentration binds to
many different proteins at a constant weight-weight ratio there is a defined number of bound SDS charges per amino acid residue therefore the charge contributed by SDS is proportional to protein molecular weight
the complexes between SDS and proteins are extended structures molecules with both charge and friction proportional to molecular weight As in that case this predicts that the free mobility will be essentially independent of molecular weight and that separation will be by the effect of sieving
Analysis of multisubunit structures by SDS gel electrophoresis As in the case of DNA
there exists for each particular gel type and concentration an approximately linear relationship between the logarithm of the protein molecular weight and mobility
logUri = logUrio - kiC
DNA samples (open squares) are restriction fragments from a bacterial plasmid Proteins are polymers of the globular domain of histone H5 cross- linked with either glutaraldehyde (squares) or dithiobis (succinimidyl propionate) (circles)
References
Section 54 in Principles of Physical Biochemistry
ELECTROPHORESIS
electric charge differences can be used to separate and analyze mixtures of biopolymers electrophoretic methods are used in every area of biochemistry and molecular charged most important physical technique available to scientists working in these fields
Electrophoresis General Principles Electrophoresis The transport of particles by
an electrical field The charged molecule is not alone but in the
presence of many other charged particles and these will both influence the local field and interact with the macromolecule making analysis difficult
Diffusion through a solvent
The Diffusion coefficient and the fraction coefficient Diffusion coefficient introduced Albert Einstein
D = kTf
f friction coefficient-measures the resistance encountered by the molecule in moving through the solvent
Stokesrsquo Law for a sphere of radius a
fo = 6a
the viscosity of the solvent
Do = kT6a
Electrophoresis General Principles An idealized simplified situation an isolated charged
particle in a nonconducting medium
The force experienced by a particle in an electrical field is given by Coulombrsquos law
F = ZeE (E-electric field potential per unit length) The viscous resistance of the medium to the motion -
fv (f the frictional factor) The viscous resistance of the medium just balances
the driving force fv = F = ZeE
Electrophoresis General Principles
Electrophoretic mobility U (the ratio of velocity to the strength of the driving field)
U = vE = Zef If the particle happens to be spherical Stokesrsquos law
applies U = Ze6a
The zonal techniques In these methods a thin layer or zone of the macromolecule solution is electrophoresed through some kind of matrix
The matrix provides stability against convection In addition in many cases the matrix acts as a molecular sieve to aid in the separation of molecules on the basis of size
Electrophoresis General Principles The kind of supporting matrix used depends on the type of
molecules to be separated and on the desired basis for separation charge molecular weight or both
Almost all electrophoresis of biological macromolecules is at
present carried out on either polyacrylamide or agarose gels
Electrophoresis
Each macromolecular solution is applied in a thin layer in one well
If several components of different mobility are present they will separate during electrophoresis just as the zones of molecules of different sedimentation rate separate in zonal centrifugation
Usually a dye of high mobility is added its migration serves to mark the progress of the experiment
Electrophoresis
The dye also serves as a convenient measure of mobility the relative mobility Uri of each component i is defined by
Ud is the dye mobility and di and dd are the distances that component i and dye respectively have moved by the conclusion of the experiment
Ferguson plots
A very simple relationship between relative mobility and gel concentration
logUri = logUrio - kiC
where C is the gel concentration and is the relative mobility of component i when C = 0 that is the relative mobility in free electrophoresis The constant ki large molecules will have large values of k whereas very small molecules will have small values and hence will behave almost the same way in a gel as they do in free electrophoresis
Electrophoresis
Ferguson plots for a number of commonly encountered solutions
Example
Analysis of multisubunit structures by SDS gel electrophoresis a very widely used technique for the estimation of the
molecular weights of polypeptide chains the protein to be studied is first heated in a dilute
solution of a detergent such as sodium dodecyl sulfate (SDS) This breaks down all native quaternary tertiary and secondary structures in the protein Usually a reducing agent such as -mercaptoethanol is also
added to reduce any disulfide bonds The protein is then electrophoresed in the presence of SDS The separation proceeds on the basis of polypeptide-chain
weight and is nearly independent of the charge on the polypeptide
Analysis of multisubunit structures by SDS gel electrophoresisGeneral principles First SDS at a given solution concentration binds to
many different proteins at a constant weight-weight ratio there is a defined number of bound SDS charges per amino acid residue therefore the charge contributed by SDS is proportional to protein molecular weight
the complexes between SDS and proteins are extended structures molecules with both charge and friction proportional to molecular weight As in that case this predicts that the free mobility will be essentially independent of molecular weight and that separation will be by the effect of sieving
Analysis of multisubunit structures by SDS gel electrophoresis As in the case of DNA
there exists for each particular gel type and concentration an approximately linear relationship between the logarithm of the protein molecular weight and mobility
logUri = logUrio - kiC
DNA samples (open squares) are restriction fragments from a bacterial plasmid Proteins are polymers of the globular domain of histone H5 cross- linked with either glutaraldehyde (squares) or dithiobis (succinimidyl propionate) (circles)
References
Section 54 in Principles of Physical Biochemistry
Electrophoresis General Principles Electrophoresis The transport of particles by
an electrical field The charged molecule is not alone but in the
presence of many other charged particles and these will both influence the local field and interact with the macromolecule making analysis difficult
Diffusion through a solvent
The Diffusion coefficient and the fraction coefficient Diffusion coefficient introduced Albert Einstein
D = kTf
f friction coefficient-measures the resistance encountered by the molecule in moving through the solvent
Stokesrsquo Law for a sphere of radius a
fo = 6a
the viscosity of the solvent
Do = kT6a
Electrophoresis General Principles An idealized simplified situation an isolated charged
particle in a nonconducting medium
The force experienced by a particle in an electrical field is given by Coulombrsquos law
F = ZeE (E-electric field potential per unit length) The viscous resistance of the medium to the motion -
fv (f the frictional factor) The viscous resistance of the medium just balances
the driving force fv = F = ZeE
Electrophoresis General Principles
Electrophoretic mobility U (the ratio of velocity to the strength of the driving field)
U = vE = Zef If the particle happens to be spherical Stokesrsquos law
applies U = Ze6a
The zonal techniques In these methods a thin layer or zone of the macromolecule solution is electrophoresed through some kind of matrix
The matrix provides stability against convection In addition in many cases the matrix acts as a molecular sieve to aid in the separation of molecules on the basis of size
Electrophoresis General Principles The kind of supporting matrix used depends on the type of
molecules to be separated and on the desired basis for separation charge molecular weight or both
Almost all electrophoresis of biological macromolecules is at
present carried out on either polyacrylamide or agarose gels
Electrophoresis
Each macromolecular solution is applied in a thin layer in one well
If several components of different mobility are present they will separate during electrophoresis just as the zones of molecules of different sedimentation rate separate in zonal centrifugation
Usually a dye of high mobility is added its migration serves to mark the progress of the experiment
Electrophoresis
The dye also serves as a convenient measure of mobility the relative mobility Uri of each component i is defined by
Ud is the dye mobility and di and dd are the distances that component i and dye respectively have moved by the conclusion of the experiment
Ferguson plots
A very simple relationship between relative mobility and gel concentration
logUri = logUrio - kiC
where C is the gel concentration and is the relative mobility of component i when C = 0 that is the relative mobility in free electrophoresis The constant ki large molecules will have large values of k whereas very small molecules will have small values and hence will behave almost the same way in a gel as they do in free electrophoresis
Electrophoresis
Ferguson plots for a number of commonly encountered solutions
Example
Analysis of multisubunit structures by SDS gel electrophoresis a very widely used technique for the estimation of the
molecular weights of polypeptide chains the protein to be studied is first heated in a dilute
solution of a detergent such as sodium dodecyl sulfate (SDS) This breaks down all native quaternary tertiary and secondary structures in the protein Usually a reducing agent such as -mercaptoethanol is also
added to reduce any disulfide bonds The protein is then electrophoresed in the presence of SDS The separation proceeds on the basis of polypeptide-chain
weight and is nearly independent of the charge on the polypeptide
Analysis of multisubunit structures by SDS gel electrophoresisGeneral principles First SDS at a given solution concentration binds to
many different proteins at a constant weight-weight ratio there is a defined number of bound SDS charges per amino acid residue therefore the charge contributed by SDS is proportional to protein molecular weight
the complexes between SDS and proteins are extended structures molecules with both charge and friction proportional to molecular weight As in that case this predicts that the free mobility will be essentially independent of molecular weight and that separation will be by the effect of sieving
Analysis of multisubunit structures by SDS gel electrophoresis As in the case of DNA
there exists for each particular gel type and concentration an approximately linear relationship between the logarithm of the protein molecular weight and mobility
logUri = logUrio - kiC
DNA samples (open squares) are restriction fragments from a bacterial plasmid Proteins are polymers of the globular domain of histone H5 cross- linked with either glutaraldehyde (squares) or dithiobis (succinimidyl propionate) (circles)
References
Section 54 in Principles of Physical Biochemistry
Diffusion through a solvent
The Diffusion coefficient and the fraction coefficient Diffusion coefficient introduced Albert Einstein
D = kTf
f friction coefficient-measures the resistance encountered by the molecule in moving through the solvent
Stokesrsquo Law for a sphere of radius a
fo = 6a
the viscosity of the solvent
Do = kT6a
Electrophoresis General Principles An idealized simplified situation an isolated charged
particle in a nonconducting medium
The force experienced by a particle in an electrical field is given by Coulombrsquos law
F = ZeE (E-electric field potential per unit length) The viscous resistance of the medium to the motion -
fv (f the frictional factor) The viscous resistance of the medium just balances
the driving force fv = F = ZeE
Electrophoresis General Principles
Electrophoretic mobility U (the ratio of velocity to the strength of the driving field)
U = vE = Zef If the particle happens to be spherical Stokesrsquos law
applies U = Ze6a
The zonal techniques In these methods a thin layer or zone of the macromolecule solution is electrophoresed through some kind of matrix
The matrix provides stability against convection In addition in many cases the matrix acts as a molecular sieve to aid in the separation of molecules on the basis of size
Electrophoresis General Principles The kind of supporting matrix used depends on the type of
molecules to be separated and on the desired basis for separation charge molecular weight or both
Almost all electrophoresis of biological macromolecules is at
present carried out on either polyacrylamide or agarose gels
Electrophoresis
Each macromolecular solution is applied in a thin layer in one well
If several components of different mobility are present they will separate during electrophoresis just as the zones of molecules of different sedimentation rate separate in zonal centrifugation
Usually a dye of high mobility is added its migration serves to mark the progress of the experiment
Electrophoresis
The dye also serves as a convenient measure of mobility the relative mobility Uri of each component i is defined by
Ud is the dye mobility and di and dd are the distances that component i and dye respectively have moved by the conclusion of the experiment
Ferguson plots
A very simple relationship between relative mobility and gel concentration
logUri = logUrio - kiC
where C is the gel concentration and is the relative mobility of component i when C = 0 that is the relative mobility in free electrophoresis The constant ki large molecules will have large values of k whereas very small molecules will have small values and hence will behave almost the same way in a gel as they do in free electrophoresis
Electrophoresis
Ferguson plots for a number of commonly encountered solutions
Example
Analysis of multisubunit structures by SDS gel electrophoresis a very widely used technique for the estimation of the
molecular weights of polypeptide chains the protein to be studied is first heated in a dilute
solution of a detergent such as sodium dodecyl sulfate (SDS) This breaks down all native quaternary tertiary and secondary structures in the protein Usually a reducing agent such as -mercaptoethanol is also
added to reduce any disulfide bonds The protein is then electrophoresed in the presence of SDS The separation proceeds on the basis of polypeptide-chain
weight and is nearly independent of the charge on the polypeptide
Analysis of multisubunit structures by SDS gel electrophoresisGeneral principles First SDS at a given solution concentration binds to
many different proteins at a constant weight-weight ratio there is a defined number of bound SDS charges per amino acid residue therefore the charge contributed by SDS is proportional to protein molecular weight
the complexes between SDS and proteins are extended structures molecules with both charge and friction proportional to molecular weight As in that case this predicts that the free mobility will be essentially independent of molecular weight and that separation will be by the effect of sieving
Analysis of multisubunit structures by SDS gel electrophoresis As in the case of DNA
there exists for each particular gel type and concentration an approximately linear relationship between the logarithm of the protein molecular weight and mobility
logUri = logUrio - kiC
DNA samples (open squares) are restriction fragments from a bacterial plasmid Proteins are polymers of the globular domain of histone H5 cross- linked with either glutaraldehyde (squares) or dithiobis (succinimidyl propionate) (circles)
References
Section 54 in Principles of Physical Biochemistry
Electrophoresis General Principles An idealized simplified situation an isolated charged
particle in a nonconducting medium
The force experienced by a particle in an electrical field is given by Coulombrsquos law
F = ZeE (E-electric field potential per unit length) The viscous resistance of the medium to the motion -
fv (f the frictional factor) The viscous resistance of the medium just balances
the driving force fv = F = ZeE
Electrophoresis General Principles
Electrophoretic mobility U (the ratio of velocity to the strength of the driving field)
U = vE = Zef If the particle happens to be spherical Stokesrsquos law
applies U = Ze6a
The zonal techniques In these methods a thin layer or zone of the macromolecule solution is electrophoresed through some kind of matrix
The matrix provides stability against convection In addition in many cases the matrix acts as a molecular sieve to aid in the separation of molecules on the basis of size
Electrophoresis General Principles The kind of supporting matrix used depends on the type of
molecules to be separated and on the desired basis for separation charge molecular weight or both
Almost all electrophoresis of biological macromolecules is at
present carried out on either polyacrylamide or agarose gels
Electrophoresis
Each macromolecular solution is applied in a thin layer in one well
If several components of different mobility are present they will separate during electrophoresis just as the zones of molecules of different sedimentation rate separate in zonal centrifugation
Usually a dye of high mobility is added its migration serves to mark the progress of the experiment
Electrophoresis
The dye also serves as a convenient measure of mobility the relative mobility Uri of each component i is defined by
Ud is the dye mobility and di and dd are the distances that component i and dye respectively have moved by the conclusion of the experiment
Ferguson plots
A very simple relationship between relative mobility and gel concentration
logUri = logUrio - kiC
where C is the gel concentration and is the relative mobility of component i when C = 0 that is the relative mobility in free electrophoresis The constant ki large molecules will have large values of k whereas very small molecules will have small values and hence will behave almost the same way in a gel as they do in free electrophoresis
Electrophoresis
Ferguson plots for a number of commonly encountered solutions
Example
Analysis of multisubunit structures by SDS gel electrophoresis a very widely used technique for the estimation of the
molecular weights of polypeptide chains the protein to be studied is first heated in a dilute
solution of a detergent such as sodium dodecyl sulfate (SDS) This breaks down all native quaternary tertiary and secondary structures in the protein Usually a reducing agent such as -mercaptoethanol is also
added to reduce any disulfide bonds The protein is then electrophoresed in the presence of SDS The separation proceeds on the basis of polypeptide-chain
weight and is nearly independent of the charge on the polypeptide
Analysis of multisubunit structures by SDS gel electrophoresisGeneral principles First SDS at a given solution concentration binds to
many different proteins at a constant weight-weight ratio there is a defined number of bound SDS charges per amino acid residue therefore the charge contributed by SDS is proportional to protein molecular weight
the complexes between SDS and proteins are extended structures molecules with both charge and friction proportional to molecular weight As in that case this predicts that the free mobility will be essentially independent of molecular weight and that separation will be by the effect of sieving
Analysis of multisubunit structures by SDS gel electrophoresis As in the case of DNA
there exists for each particular gel type and concentration an approximately linear relationship between the logarithm of the protein molecular weight and mobility
logUri = logUrio - kiC
DNA samples (open squares) are restriction fragments from a bacterial plasmid Proteins are polymers of the globular domain of histone H5 cross- linked with either glutaraldehyde (squares) or dithiobis (succinimidyl propionate) (circles)
References
Section 54 in Principles of Physical Biochemistry
Electrophoresis General Principles
Electrophoretic mobility U (the ratio of velocity to the strength of the driving field)
U = vE = Zef If the particle happens to be spherical Stokesrsquos law
applies U = Ze6a
The zonal techniques In these methods a thin layer or zone of the macromolecule solution is electrophoresed through some kind of matrix
The matrix provides stability against convection In addition in many cases the matrix acts as a molecular sieve to aid in the separation of molecules on the basis of size
Electrophoresis General Principles The kind of supporting matrix used depends on the type of
molecules to be separated and on the desired basis for separation charge molecular weight or both
Almost all electrophoresis of biological macromolecules is at
present carried out on either polyacrylamide or agarose gels
Electrophoresis
Each macromolecular solution is applied in a thin layer in one well
If several components of different mobility are present they will separate during electrophoresis just as the zones of molecules of different sedimentation rate separate in zonal centrifugation
Usually a dye of high mobility is added its migration serves to mark the progress of the experiment
Electrophoresis
The dye also serves as a convenient measure of mobility the relative mobility Uri of each component i is defined by
Ud is the dye mobility and di and dd are the distances that component i and dye respectively have moved by the conclusion of the experiment
Ferguson plots
A very simple relationship between relative mobility and gel concentration
logUri = logUrio - kiC
where C is the gel concentration and is the relative mobility of component i when C = 0 that is the relative mobility in free electrophoresis The constant ki large molecules will have large values of k whereas very small molecules will have small values and hence will behave almost the same way in a gel as they do in free electrophoresis
Electrophoresis
Ferguson plots for a number of commonly encountered solutions
Example
Analysis of multisubunit structures by SDS gel electrophoresis a very widely used technique for the estimation of the
molecular weights of polypeptide chains the protein to be studied is first heated in a dilute
solution of a detergent such as sodium dodecyl sulfate (SDS) This breaks down all native quaternary tertiary and secondary structures in the protein Usually a reducing agent such as -mercaptoethanol is also
added to reduce any disulfide bonds The protein is then electrophoresed in the presence of SDS The separation proceeds on the basis of polypeptide-chain
weight and is nearly independent of the charge on the polypeptide
Analysis of multisubunit structures by SDS gel electrophoresisGeneral principles First SDS at a given solution concentration binds to
many different proteins at a constant weight-weight ratio there is a defined number of bound SDS charges per amino acid residue therefore the charge contributed by SDS is proportional to protein molecular weight
the complexes between SDS and proteins are extended structures molecules with both charge and friction proportional to molecular weight As in that case this predicts that the free mobility will be essentially independent of molecular weight and that separation will be by the effect of sieving
Analysis of multisubunit structures by SDS gel electrophoresis As in the case of DNA
there exists for each particular gel type and concentration an approximately linear relationship between the logarithm of the protein molecular weight and mobility
logUri = logUrio - kiC
DNA samples (open squares) are restriction fragments from a bacterial plasmid Proteins are polymers of the globular domain of histone H5 cross- linked with either glutaraldehyde (squares) or dithiobis (succinimidyl propionate) (circles)
References
Section 54 in Principles of Physical Biochemistry
Electrophoresis General Principles The kind of supporting matrix used depends on the type of
molecules to be separated and on the desired basis for separation charge molecular weight or both
Almost all electrophoresis of biological macromolecules is at
present carried out on either polyacrylamide or agarose gels
Electrophoresis
Each macromolecular solution is applied in a thin layer in one well
If several components of different mobility are present they will separate during electrophoresis just as the zones of molecules of different sedimentation rate separate in zonal centrifugation
Usually a dye of high mobility is added its migration serves to mark the progress of the experiment
Electrophoresis
The dye also serves as a convenient measure of mobility the relative mobility Uri of each component i is defined by
Ud is the dye mobility and di and dd are the distances that component i and dye respectively have moved by the conclusion of the experiment
Ferguson plots
A very simple relationship between relative mobility and gel concentration
logUri = logUrio - kiC
where C is the gel concentration and is the relative mobility of component i when C = 0 that is the relative mobility in free electrophoresis The constant ki large molecules will have large values of k whereas very small molecules will have small values and hence will behave almost the same way in a gel as they do in free electrophoresis
Electrophoresis
Ferguson plots for a number of commonly encountered solutions
Example
Analysis of multisubunit structures by SDS gel electrophoresis a very widely used technique for the estimation of the
molecular weights of polypeptide chains the protein to be studied is first heated in a dilute
solution of a detergent such as sodium dodecyl sulfate (SDS) This breaks down all native quaternary tertiary and secondary structures in the protein Usually a reducing agent such as -mercaptoethanol is also
added to reduce any disulfide bonds The protein is then electrophoresed in the presence of SDS The separation proceeds on the basis of polypeptide-chain
weight and is nearly independent of the charge on the polypeptide
Analysis of multisubunit structures by SDS gel electrophoresisGeneral principles First SDS at a given solution concentration binds to
many different proteins at a constant weight-weight ratio there is a defined number of bound SDS charges per amino acid residue therefore the charge contributed by SDS is proportional to protein molecular weight
the complexes between SDS and proteins are extended structures molecules with both charge and friction proportional to molecular weight As in that case this predicts that the free mobility will be essentially independent of molecular weight and that separation will be by the effect of sieving
Analysis of multisubunit structures by SDS gel electrophoresis As in the case of DNA
there exists for each particular gel type and concentration an approximately linear relationship between the logarithm of the protein molecular weight and mobility
logUri = logUrio - kiC
DNA samples (open squares) are restriction fragments from a bacterial plasmid Proteins are polymers of the globular domain of histone H5 cross- linked with either glutaraldehyde (squares) or dithiobis (succinimidyl propionate) (circles)
References
Section 54 in Principles of Physical Biochemistry
Electrophoresis
Each macromolecular solution is applied in a thin layer in one well
If several components of different mobility are present they will separate during electrophoresis just as the zones of molecules of different sedimentation rate separate in zonal centrifugation
Usually a dye of high mobility is added its migration serves to mark the progress of the experiment
Electrophoresis
The dye also serves as a convenient measure of mobility the relative mobility Uri of each component i is defined by
Ud is the dye mobility and di and dd are the distances that component i and dye respectively have moved by the conclusion of the experiment
Ferguson plots
A very simple relationship between relative mobility and gel concentration
logUri = logUrio - kiC
where C is the gel concentration and is the relative mobility of component i when C = 0 that is the relative mobility in free electrophoresis The constant ki large molecules will have large values of k whereas very small molecules will have small values and hence will behave almost the same way in a gel as they do in free electrophoresis
Electrophoresis
Ferguson plots for a number of commonly encountered solutions
Example
Analysis of multisubunit structures by SDS gel electrophoresis a very widely used technique for the estimation of the
molecular weights of polypeptide chains the protein to be studied is first heated in a dilute
solution of a detergent such as sodium dodecyl sulfate (SDS) This breaks down all native quaternary tertiary and secondary structures in the protein Usually a reducing agent such as -mercaptoethanol is also
added to reduce any disulfide bonds The protein is then electrophoresed in the presence of SDS The separation proceeds on the basis of polypeptide-chain
weight and is nearly independent of the charge on the polypeptide
Analysis of multisubunit structures by SDS gel electrophoresisGeneral principles First SDS at a given solution concentration binds to
many different proteins at a constant weight-weight ratio there is a defined number of bound SDS charges per amino acid residue therefore the charge contributed by SDS is proportional to protein molecular weight
the complexes between SDS and proteins are extended structures molecules with both charge and friction proportional to molecular weight As in that case this predicts that the free mobility will be essentially independent of molecular weight and that separation will be by the effect of sieving
Analysis of multisubunit structures by SDS gel electrophoresis As in the case of DNA
there exists for each particular gel type and concentration an approximately linear relationship between the logarithm of the protein molecular weight and mobility
logUri = logUrio - kiC
DNA samples (open squares) are restriction fragments from a bacterial plasmid Proteins are polymers of the globular domain of histone H5 cross- linked with either glutaraldehyde (squares) or dithiobis (succinimidyl propionate) (circles)
References
Section 54 in Principles of Physical Biochemistry
Electrophoresis
The dye also serves as a convenient measure of mobility the relative mobility Uri of each component i is defined by
Ud is the dye mobility and di and dd are the distances that component i and dye respectively have moved by the conclusion of the experiment
Ferguson plots
A very simple relationship between relative mobility and gel concentration
logUri = logUrio - kiC
where C is the gel concentration and is the relative mobility of component i when C = 0 that is the relative mobility in free electrophoresis The constant ki large molecules will have large values of k whereas very small molecules will have small values and hence will behave almost the same way in a gel as they do in free electrophoresis
Electrophoresis
Ferguson plots for a number of commonly encountered solutions
Example
Analysis of multisubunit structures by SDS gel electrophoresis a very widely used technique for the estimation of the
molecular weights of polypeptide chains the protein to be studied is first heated in a dilute
solution of a detergent such as sodium dodecyl sulfate (SDS) This breaks down all native quaternary tertiary and secondary structures in the protein Usually a reducing agent such as -mercaptoethanol is also
added to reduce any disulfide bonds The protein is then electrophoresed in the presence of SDS The separation proceeds on the basis of polypeptide-chain
weight and is nearly independent of the charge on the polypeptide
Analysis of multisubunit structures by SDS gel electrophoresisGeneral principles First SDS at a given solution concentration binds to
many different proteins at a constant weight-weight ratio there is a defined number of bound SDS charges per amino acid residue therefore the charge contributed by SDS is proportional to protein molecular weight
the complexes between SDS and proteins are extended structures molecules with both charge and friction proportional to molecular weight As in that case this predicts that the free mobility will be essentially independent of molecular weight and that separation will be by the effect of sieving
Analysis of multisubunit structures by SDS gel electrophoresis As in the case of DNA
there exists for each particular gel type and concentration an approximately linear relationship between the logarithm of the protein molecular weight and mobility
logUri = logUrio - kiC
DNA samples (open squares) are restriction fragments from a bacterial plasmid Proteins are polymers of the globular domain of histone H5 cross- linked with either glutaraldehyde (squares) or dithiobis (succinimidyl propionate) (circles)
References
Section 54 in Principles of Physical Biochemistry
Ferguson plots
A very simple relationship between relative mobility and gel concentration
logUri = logUrio - kiC
where C is the gel concentration and is the relative mobility of component i when C = 0 that is the relative mobility in free electrophoresis The constant ki large molecules will have large values of k whereas very small molecules will have small values and hence will behave almost the same way in a gel as they do in free electrophoresis
Electrophoresis
Ferguson plots for a number of commonly encountered solutions
Example
Analysis of multisubunit structures by SDS gel electrophoresis a very widely used technique for the estimation of the
molecular weights of polypeptide chains the protein to be studied is first heated in a dilute
solution of a detergent such as sodium dodecyl sulfate (SDS) This breaks down all native quaternary tertiary and secondary structures in the protein Usually a reducing agent such as -mercaptoethanol is also
added to reduce any disulfide bonds The protein is then electrophoresed in the presence of SDS The separation proceeds on the basis of polypeptide-chain
weight and is nearly independent of the charge on the polypeptide
Analysis of multisubunit structures by SDS gel electrophoresisGeneral principles First SDS at a given solution concentration binds to
many different proteins at a constant weight-weight ratio there is a defined number of bound SDS charges per amino acid residue therefore the charge contributed by SDS is proportional to protein molecular weight
the complexes between SDS and proteins are extended structures molecules with both charge and friction proportional to molecular weight As in that case this predicts that the free mobility will be essentially independent of molecular weight and that separation will be by the effect of sieving
Analysis of multisubunit structures by SDS gel electrophoresis As in the case of DNA
there exists for each particular gel type and concentration an approximately linear relationship between the logarithm of the protein molecular weight and mobility
logUri = logUrio - kiC
DNA samples (open squares) are restriction fragments from a bacterial plasmid Proteins are polymers of the globular domain of histone H5 cross- linked with either glutaraldehyde (squares) or dithiobis (succinimidyl propionate) (circles)
References
Section 54 in Principles of Physical Biochemistry
Electrophoresis
Ferguson plots for a number of commonly encountered solutions
Example
Analysis of multisubunit structures by SDS gel electrophoresis a very widely used technique for the estimation of the
molecular weights of polypeptide chains the protein to be studied is first heated in a dilute
solution of a detergent such as sodium dodecyl sulfate (SDS) This breaks down all native quaternary tertiary and secondary structures in the protein Usually a reducing agent such as -mercaptoethanol is also
added to reduce any disulfide bonds The protein is then electrophoresed in the presence of SDS The separation proceeds on the basis of polypeptide-chain
weight and is nearly independent of the charge on the polypeptide
Analysis of multisubunit structures by SDS gel electrophoresisGeneral principles First SDS at a given solution concentration binds to
many different proteins at a constant weight-weight ratio there is a defined number of bound SDS charges per amino acid residue therefore the charge contributed by SDS is proportional to protein molecular weight
the complexes between SDS and proteins are extended structures molecules with both charge and friction proportional to molecular weight As in that case this predicts that the free mobility will be essentially independent of molecular weight and that separation will be by the effect of sieving
Analysis of multisubunit structures by SDS gel electrophoresis As in the case of DNA
there exists for each particular gel type and concentration an approximately linear relationship between the logarithm of the protein molecular weight and mobility
logUri = logUrio - kiC
DNA samples (open squares) are restriction fragments from a bacterial plasmid Proteins are polymers of the globular domain of histone H5 cross- linked with either glutaraldehyde (squares) or dithiobis (succinimidyl propionate) (circles)
References
Section 54 in Principles of Physical Biochemistry
Example
Analysis of multisubunit structures by SDS gel electrophoresis a very widely used technique for the estimation of the
molecular weights of polypeptide chains the protein to be studied is first heated in a dilute
solution of a detergent such as sodium dodecyl sulfate (SDS) This breaks down all native quaternary tertiary and secondary structures in the protein Usually a reducing agent such as -mercaptoethanol is also
added to reduce any disulfide bonds The protein is then electrophoresed in the presence of SDS The separation proceeds on the basis of polypeptide-chain
weight and is nearly independent of the charge on the polypeptide
Analysis of multisubunit structures by SDS gel electrophoresisGeneral principles First SDS at a given solution concentration binds to
many different proteins at a constant weight-weight ratio there is a defined number of bound SDS charges per amino acid residue therefore the charge contributed by SDS is proportional to protein molecular weight
the complexes between SDS and proteins are extended structures molecules with both charge and friction proportional to molecular weight As in that case this predicts that the free mobility will be essentially independent of molecular weight and that separation will be by the effect of sieving
Analysis of multisubunit structures by SDS gel electrophoresis As in the case of DNA
there exists for each particular gel type and concentration an approximately linear relationship between the logarithm of the protein molecular weight and mobility
logUri = logUrio - kiC
DNA samples (open squares) are restriction fragments from a bacterial plasmid Proteins are polymers of the globular domain of histone H5 cross- linked with either glutaraldehyde (squares) or dithiobis (succinimidyl propionate) (circles)
References
Section 54 in Principles of Physical Biochemistry
Analysis of multisubunit structures by SDS gel electrophoresis a very widely used technique for the estimation of the
molecular weights of polypeptide chains the protein to be studied is first heated in a dilute
solution of a detergent such as sodium dodecyl sulfate (SDS) This breaks down all native quaternary tertiary and secondary structures in the protein Usually a reducing agent such as -mercaptoethanol is also
added to reduce any disulfide bonds The protein is then electrophoresed in the presence of SDS The separation proceeds on the basis of polypeptide-chain
weight and is nearly independent of the charge on the polypeptide
Analysis of multisubunit structures by SDS gel electrophoresisGeneral principles First SDS at a given solution concentration binds to
many different proteins at a constant weight-weight ratio there is a defined number of bound SDS charges per amino acid residue therefore the charge contributed by SDS is proportional to protein molecular weight
the complexes between SDS and proteins are extended structures molecules with both charge and friction proportional to molecular weight As in that case this predicts that the free mobility will be essentially independent of molecular weight and that separation will be by the effect of sieving
Analysis of multisubunit structures by SDS gel electrophoresis As in the case of DNA
there exists for each particular gel type and concentration an approximately linear relationship between the logarithm of the protein molecular weight and mobility
logUri = logUrio - kiC
DNA samples (open squares) are restriction fragments from a bacterial plasmid Proteins are polymers of the globular domain of histone H5 cross- linked with either glutaraldehyde (squares) or dithiobis (succinimidyl propionate) (circles)
References
Section 54 in Principles of Physical Biochemistry
Analysis of multisubunit structures by SDS gel electrophoresisGeneral principles First SDS at a given solution concentration binds to
many different proteins at a constant weight-weight ratio there is a defined number of bound SDS charges per amino acid residue therefore the charge contributed by SDS is proportional to protein molecular weight
the complexes between SDS and proteins are extended structures molecules with both charge and friction proportional to molecular weight As in that case this predicts that the free mobility will be essentially independent of molecular weight and that separation will be by the effect of sieving
Analysis of multisubunit structures by SDS gel electrophoresis As in the case of DNA
there exists for each particular gel type and concentration an approximately linear relationship between the logarithm of the protein molecular weight and mobility
logUri = logUrio - kiC
DNA samples (open squares) are restriction fragments from a bacterial plasmid Proteins are polymers of the globular domain of histone H5 cross- linked with either glutaraldehyde (squares) or dithiobis (succinimidyl propionate) (circles)
References
Section 54 in Principles of Physical Biochemistry
Analysis of multisubunit structures by SDS gel electrophoresis As in the case of DNA
there exists for each particular gel type and concentration an approximately linear relationship between the logarithm of the protein molecular weight and mobility
logUri = logUrio - kiC
DNA samples (open squares) are restriction fragments from a bacterial plasmid Proteins are polymers of the globular domain of histone H5 cross- linked with either glutaraldehyde (squares) or dithiobis (succinimidyl propionate) (circles)
References
Section 54 in Principles of Physical Biochemistry
References
Section 54 in Principles of Physical Biochemistry