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TECHNIQUES IN TECHNIQUES IN MOLECULAR BIOLOGYMOLECULAR BIOLOGY
• CENTRIFUGATION- Separation of molecules/macromolecules/organelles according to the size, shape, density & gradient
• ELECTROPHORESIS- Separation of molecules/macromolecules according to charge
• MICROSCOPY- Structural examination of minute molecule/macromolecule/organelle
CENTRIFUGATIONCENTRIFUGATION
• MATERIALS OR PARTICLES IN A SOLUTION CAN BE SEPARATED BY A CENTRIFUGE THAT USES THE PRINCIPLE OF CENTRIFUGATION
• CLASSES:
-ANALYTICAL/PREPARATIVE
-ULTRACENTRIFUGATION AND LOW SPEED
-DIFFERENTIAL/ZONAL CENTRIFUGATION
http://ntri.tamuk.edu/centrifuge/centrifugation.html
ANALYTICAL CENTRIFUGATIONANALYTICAL CENTRIFUGATION
• IS USED TO MEASURE THE SEDIMENTED PARTICLE PHYSICAL CHARACTERISTICS SUCH AS SEDIMENTATION COEFFICIENT AND MOLECULAR WEIGHT
PREPARATIVE PREPARATIVE CENTRIFUGATIONCENTRIFUGATION
• TO SEPARATE SPECIFIC PARTICLES THAT IS REUSABLE
• TYPES:
- RATE ZONAL
- DIFFERENTIAL
- ISOPYCNIC CENTRIFUGATION
ULTRACENTRIFUGATION AND ULTRACENTRIFUGATION AND LOW SPEEDLOW SPEED
• DEPENDS ON SPEED• ULTRACENTRIFUGATION - THE SPEED
EXCEEDS 20,000 RPM• SUPER SPEED ULTRACENTRIFUGATION-
THE SPEED IS BETWEEN 10,000 RPM-20,000 RPM
• LOW SPEED CENTRIFUGATION- THE SPEED IS BELOW 10,000 RPM
DIFFERENTIAL DIFFERENTIAL CENTRIFUGATIONCENTRIFUGATION
• PARTICLES IN SAMPLE WILL SEPARATE INTO SUPERNATANT AND PELLET OR IN BOTH DEPENDING ON THEIR SIZE, SHAPE, DENSITY AND CENTRIFUGATION CONDITION
• THE PELLET CONTAINS ALL THE SEDIMENTED COMPONENT MIXTURE AND CAN CONTAIN MATERIALS THAT WAS NOT SEDIMENTED EARLIER
DIFFERENTIAL DIFFERENTIAL CENTRIFUGATIONCENTRIFUGATION
• SUPERNATANT CONTAINS MATERIALS THAT ARE NOT SEDIMENTED BUT CAN BE SEDIMENTED WHEN CENTRIFUGATION IS DONE AT A HIGHER SPEED
DIFFERENTIAL DIFFERENTIAL CENTRIFUGATIONCENTRIFUGATION
ZONAL CENTRIFUGATIONZONAL CENTRIFUGATION
• SAMPLE IS APPLIED ON TOP OF SUCROSE OR CESIUM CLORIDE SOLUTION
• PARTICLE CAN BE SEPARATED ACCORDING TO SIZE & SHAPE (TIME-RATE ZONE) OR DENSITY (ISOPYCNIC)
RATE-ZONAL RATE-ZONAL CENTRIFUGATIONCENTRIFUGATION
ISOPYCNICISOPYCNIC--ZONAL ZONAL CENTRIFUGATIONCENTRIFUGATION
SEDIMENTATION COEFFICIENTSEDIMENTATION COEFFICIENT
• WHEN CELL COMPONENTS ARE CENTRIFUFED THROUGH A GRADIENT SOLUTION, THEY WILL SEPARATE INTO THEIR OWN ZONE OR LINE/LAYER
• THE RATE WHEN THE COMPONENT SEPARATES IS CALLED AS SEDIMENTATION COEFFICIENT OR THE s VALUE (SVEDBERG UNIT )
1 S = 1 X 10-13 SECONDS
SEDIMENTATION COEFFICIENTSEDIMENTATION COEFFICIENTVALUESVALUES
PARTICLE OR SEDIMENTATION
MOLECULE COEFFICIENT
LYSOSOME 9400S
TOBACCO MOSAIC VIRUS 198S
RIBOSOME 80S
RIBOSOMAL RNA MOLECULE 28S
tRNA MOLECULE 4S
HEMOGLOBIN MOLECULE 4.5S
SPEED OF CENTRIFUGATION
• A PARTICLE THAT IS ROTATING WILL HAVE A PULLING FORCE IN A FORM OF MAGNITUDE TO SPEED FUNCTION AT DEFINED ANGLE (ROTATION SPEED) AND CENTRFUGATION RADIUS (THE DISTANCE BETWEEN THE SAMPLE CONTAINER AND THE ROTOR CENTRE)
SPEED OF CENTRIFUGATION
• 2 WAYS OF EXPRESSING THE PULLING FORCE:
a) RELATIVE CENTRIFUGATIONAL FORCE-RCF (g)
b) ROTATION PER MINUTE (rpm)
RELATIVE RELATIVE CENTRIFUGATIONAL FORCECENTRIFUGATIONAL FORCE
• THE PULLING FORCE OF CENTRIFUGATION IS BASED ON OR RELATIVE TO THE STANDARD GRAVITATIONAL FORCE
• FOR EXAMPLE 500x g MEANS THAT THE PULLING FORCE IS 500 TIMES BIGGER THAN THE STANDARD GRAVITATIONAL FORCE
RELATIVE RELATIVE CENTRIFUGATIONAL FORCECENTRIFUGATIONAL FORCE
• EQUATION
R.C.F. = 1.119 x 10 -5 (rpm2) r rpm=rotation per minute
r=radius (in cm)
UNIT g
ELECTROPHORESISELECTROPHORESIS
• THE MOVEMENT OF CHARGED PARTICLE IS INFLUENCED BY ELECTRICAL CURRENT
• ELECTROPHORESIS IS THE METHOD OF SEPARATING MACROMOLECULE SUCH AS NUCLEIC ACID AND PROTEIN ACCORDING TO SIZE, ELECTRICAL CHARGE AND PHYSICAL PROPERTIES SUCH AS DENSITY ETC
• SEPARATION IS AIDED BY A MATRIX SUCH AS POLIACRYLAMIDE OR AGAROSE
ELECTROPHORESISELECTROPHORESIS• PRINCIPLE: SEPARATION OF MACROMOLECULE
DEPENDING ON TWO PROPERTIES: WEIGHT AND CHARGE
• ELECTRICAL CURRENT FROM THE ELECTRODE WILL PUSH THE MOLECULE AND AT THE SAME TIME THE OTHER ELECTRODE WILL PUT IT
• MOLECULES WILL MOVE ALONG THE PORES THAT ARE FORMED BETWEEN THE INTER-WOVEN MATRIX THAT ACTS LIKE A SIEVE TO SEAPARATE THE MOLECULE ACCORDING TO THEIR SIZE
ELECTROPHORESISELECTROPHORESIS• ELECTRICAL CURRENT WILL FORCE THE
MACROMOLECULE TO MOVE ALONG THE PORES • THE MACROMOLECULE MOVEMENT DEPENDS ON THE
ELECTRICAL FIELD FORCE, THE MOLECULE SIZE AND SHAPE, THE SAMPLE RELATIVE HYDROPHOBIC PROPERTY, IONIC STRENGTH AND THE TEMPERATURE OF THE ELECTROPHORESIS BUFFER
• DYEING WILL AID THE VISUALISATION OF MACROMOLECULE IN THE FORM OF SEPARATED SERIES OF STRIPES
PROTEIN ELECTROPHORESIS PROTEIN ELECTROPHORESIS
• PROTEIN HAS A POSITIVE OR NEGATIVE NET CHARGE AS A RESULT OF THE COMBINATION OF CHARGED AMINO ACIDS CONTAINEDIN THEM
• THE MATRIX THAT IS USUALLY USED FOR PROTEIN SEPARATION IS POLIACRYLAMIDE
• TWO DIMENSIONAL GEL ELECTROPHORESIS- PROTEIN SEPARATION ACCORDING TO ISOELECTRICAL POINTS AND MOLECULAR WEIGHT
2-D PROTEIN 2-D PROTEIN ELECTROPHORESIS ELECTROPHORESIS
• FIRST STEP/DIMENSION:
PROTEIN SEPARATION ACCORDING TO ISOELECTRIC POINT (PROTEIN CONTAINS DIFFERENT POSITIVE AND NEGATIVE CHARGE RATIO)
-ELECTROPHORESIS IS DONE ON THE GEL IN THE FORM OF TUBE; PROTEIN WILL MOVE IN A SOLUTION WITH DIFFERENT pH GRADIENT
2-D PROTEIN 2-D PROTEIN ELECTROPHORESISELECTROPHORESIS
• FIRST STEP/DIMENSION:
-PROTEIN WILL STOP WHEN IT REACHES THE pH WHICH IS EQUAL TO ITS ISOELECTRIC POINT i.e WHEN THE PROTEIN DOES NOT HAVE A NET CHARGE.
+ BASIC
- ACIDIC
2-D PROTEIN 2-D PROTEIN ELECTROPHORESISELECTROPHORESIS
• SECOND STEP/DIMENSION:• PROTEIN SEPARATION BY MOLECULAR
WEIGHT• ELECTROPHORESIS IS DONE IN AN
ORTHOGONAL DIRECTION FROM
THE FIRST STEP;
SODIUM DODECYL SULPHATE
(SDS) IS ADDED
+
-
2-D PROTEIN 2-D PROTEIN ELECTROPHORESISELECTROPHORESIS
1-D PROTEIN 1-D PROTEIN ELECTROPHORESISELECTROPHORESIS
• PROTEIN IS SEPARATED BY ITS MOLECULAR WEIGHT ONLY
• THE TECHNIQUE IS ALSO KNOWN AS POLIACRYLAMIDE GEL ELECTROPHORESIS (PAGE) OR SDS-PAGE IF SDS IS PRESENT DURING SAMPLE PREPARATION
• SIMULATION OF 1-D ELECTROPHORESIS
http://www.rit.edu/~pac8612/electro/
Electro_Sim.html
SDS-PAGESDS-PAGE• TO SEPARATE PROTEIN WITH THE SIZE OF
5 - 2,000 kDa• PORES IN BETWEEN THE POLIACRYLAMIDE
MATRIX CAN VARIES FROM 3%-30%• THE PROTEIN SAMPLE IS IN THE FORM OF
PRIMARY STRUCTURE (SAMPLE IS BOILED WITH SDS AND -MERCAPTOETHANOL PRIOR BEING LOADED ONTO GEL)
SDS-PAGESDS-PAGE• PROTEIN IS STAINED USING COOMASIE
BLUE OR SILVER• NON-DIRECTIONAL STAINING CAN BE
DONE:
-ANTIBODY BOUND WITH RADIOISOTOPE OR ENZYME, FLUORESENCE DYE
SDS-PAGESDS-PAGE• SDS FUNCTION:
NEGATIVELY CHARGED DETERGENT THAT
BINDS TO THE
HYDROPHOBIC REGION
OF THE PROTEIN
MOLECULE; AS A
RESULT THE PROTEIN BECOMES A LONG POLIPEPTIDE CHAIN AND FREE FROM OTHER PROTEINS AND LIPIDS
SDS-PAGESDS-PAGE -MERCAPTOETHANOL FUNCTION: TO
BREAK DISULPHIDE BONDS SO THAT PROTEIN SUBUNIT CAN BE ANALYSED
NUCLEIC ACID NUCLEIC ACID ELECTROPHORESISELECTROPHORESIS
• AGAROSE OR POLIACRYLAMIDE IS THE MATRIX USUALLY USED TO SEPARATE NUCLEIC ACID IN A TECHNIQUE KNOWN AS AGAROSE GEL ELECTROPHORESIS
• SAMPLE CONTAINING DNA IS LOADED INTO WELLS LOCATED NEAR TO THE NEGATIVELY CHARGED ELECTRODE
• DNA THAT IS NEGATIVELY CHARGED WILL BE ATTRACTED TO THE POSITIVE ELECTRODE
NUCLEIC ACID NUCLEIC ACID ELECTROPHORESISELECTROPHORESIS
• DNA WITH A BIGGER SIZE WILL MOVE SLOWER THAN THE SMALLER SIZE WHICH MOVE FASTER
• STAINING IS DONE USING ETHIDIUM BROMIDE (EtBr) THAT ENABLES THE VISUALISATION OF NUCLEIC ACID; EtBr IS INSERTED BETWEEN THE BASES ON THE NUCLEIC ACID
• EtBr IS ORANGE IN COLOUR WHEN LIT-UP BY ULTRA-VIOLET LIGHT
NUCLEIC ACID NUCLEIC ACID ELECTROPHORESISELECTROPHORESIS
MICROSCOPYMICROSCOPY
• ONE OF THE EARLIEST TECHNIQUE TO STUDY MACROMOLECULE
• PRINCIPLE: TO ENLARGE SMALL IMAGES • TYPES OF MICROSCOPY ACCORDING TO
THE SIZE OF IMAGE ENLARGEMENT
- LIGHT MICROSCOPE (300nm-2mm)
- ELECTRON MICROSCOPE
(0.15nm-100m)
LIGHT MICROSCOPELIGHT MICROSCOPE• IMAGE ENLARGEMENT PRINCIPLE:
LIGHT FROM BELOW OF THE MICROCOPE GOES THROUGH
THE CONDENSOR TO FOCUS THE
LIGHT TO THE SPECIMEN. • LIGHT FROM THE SPECIMEN IS
RECOLLECTED BY THE OBJECTIVE LENSE TO FORM AN IMAGE
LIGHT MICROSCOPELIGHT MICROSCOPE
• TYPES OF LIGHT MICROSCOPE :
BRIGHT-FIELD MICROSCOPE DARK-FIELD MICROSCOPE
PHASE-CONTRAST MICROSCOPE
FLUORESENCE MICROSCOPE (UV)
(FLUORESCIN/RHODAMIN)
ELECTRON MICROSCOPEELECTRON MICROSCOPE
• PRINCIPLE:
-ELECTRON IS USED (NOT LIGHT) TO ENLARGE IMAGE
-SPECIMEN MUST UNDERGO A SERIES OF PREPARATION PROCESSES SUCH AS COATING WITH THIN LAYER OF GOLD TO ALLOW EMITTED ELECTRON TO COLLIDE TO AND THEN RECOLLECTED TO FORM IMAGE ON THE SCREEN
ELECTRON MICROSCOPEELECTRON MICROSCOPE
• TYPES:
1) TRANSMISSION ELECTRON MICROSCOPE
-ELECTRON GOES THROUGH THE SPECIMEN AND IMAGE IS RECOLLECTED ON A FLUORECENS SCREEN
-THE INNER STRUCTURE OF THE SPECIMEN CAN BE SEEN
ELECTRON MICROSCOPEELECTRON MICROSCOPE
• TYPES:
2) SCANNING ELECTRON MICROSCOPE
-ELECTRON IS FOCUSSED TO THE SPECIMEN AND THEN REEMITTED (SCANNED) TO THE DETECTOR AND IMAGE IS SEND TO THE SCREEN FOR VIEWING
-THE OUTER STRUCTURE CAN BE SEEN
ELECTRON MICROSCOPEELECTRON MICROSCOPE
SCANNING ELECTRON MICROSCOPE
MOSQUITO IMAGESBY SCANNING ELECTRONMICROSCOPE
OTHER TECHNIQUESOTHER TECHNIQUES
• CHROMATOGRAPHY
-PAPER: PROTEIN SEPARATION BY USING FILTER PAPER AS THE MATRIX
-ION-EXCHANGE
-GEL FILTRATION
-AFFINITY
-HIGH PRESSURE LIQUID CHROMATOGRAPHY (HPLC)
OTHER TECHNIQUESOTHER TECHNIQUES
• RADIOISOTOPES FOR MOLECULE TAGGING : 32P, 131I, 35S, 14C, 45Ca, 3H
- RIA, ‘PULSE-CHASE’ EXPERIMENT, AUTORADIOGRAPHY
• ANTIBODY (MONOCLONE/POLYCLONE) FOR TAGGING MOLECULE: EIA, IF, ELISA
• X-RAY DIFFRACTION ANALYSIS: PROTEIN STRUCTURE DETERMINATION
• DNA RECOMBINANT TECNOLOGY