Section 2. Nucleic acids.Lesson 11. Methods of molecular biology

Lesson plan

1. Stages of research in molecular biology. 2. Preparation of samples for the study.3. Chromatography.4. Electrophoresis.5. Blotting.6. Polymerase chain reaction7. Sequencing

The student should know

1) theoretical bases of methods for sample preparation of proteins and nucleic acids for the study of their physical and chemical properties, amino acid or nucleotide sequence;

2) methods for isolation of proteins and nucleic acids from biological raw materials, protein proteolysis and DNA annealing, amplification of DNA fragments, visualization of proteins and nucleic acids, fixation, processing and storage of biological information

The student must be able to

1) explain the results of the study of proteins and nucleic acids by chromatography, electrophoresis, immuno-electrophoresis, blotting, polymerase chain reaction, sequencing

IntroductionSample preparationMethods of cell destruction (for self-study)Selection of the studied materialChromatographyElectrophoresisDialysisThe study of proteinsThe establishment of the amino acid sequence (protein sequence)Western blot proteinsResearch of nucleic acidsDNA and RNA sequencingDNA blottingBiotechnological method

Methods of molecular biology

Sequence analysis(sequel)

proteins RNA DNA

PCR

The transfer of DNA into the cells of bacteria and

eukaryotes

Transformation

Transfection

Transduction

Conjugation

Knockout and knockdown of

genes

RNA interference

CRISPR -technologies

Chemical: toluenebutanol,

antibiotics, enzymes

Stages of research in molecular biology

Sample preparation Highlighting

Proteins

RNA

DNA

Destruction of bacterial or eukaryotic cells

Protein purification: chromatography, electrophoresis,

immunoprecipitation, detection and mapping of epitopes using

monoclonal antibodies, ultrafiltration, selective deposition,

reversible denaturation

Сиквенс

PCR

Physical: RubbingUltrasonicOsmotic shockCryodestructionNitrogen explosion

Plasmid and vectors

Transfection

Find it on the Internet and write it in the technology summary

Rubbing and wiping of the cells through a sieve …

Ultrasonic cell destruction …

Osmotic shock of cells …

Multiple freeze-thawing …

Nitrogen explosion of cells …

Destruction of cells by toluene …

Destruction of cells by butanol …

Cell destruction by antibiotics …

Destruction of cells by enzymes …Protein extraction and deposition …

Methods of saltingDeposition methods

CHROMATOGRAPHY

Chromatography of proteins

Chromatography is a method of separation and determination ofsubstances based on the separation of components between two phases –mobile (eluant) and stationary (eluate, stationary phase).

The method is based on the difference in the force of interaction ofdifferent substances with the stationary phase.

Less interacting substances move faster and all substances move inthe column relative to the eluate at different speeds characteristic of thissubstance.

The stationary phase is a solid porous substance, which is called asorbent or a film of liquid deposited on a solid.

The mobile phase is a liquid or gas with the analyzed substancesdissolved in it, which flows through the stationary phase by gravity orunder pressure.

The stationary phase is pre-positioned in a chromatographic column,either as a chromatographic paper, or as a layer of aluminum oxide onaluminum foil.Substances that leave the column together with the mobile phase arecalled unstoppable, and their retention time determines the so-called"dead time" of the column.Various additional fields (gravitational, electric, magnetic, etc.) canbe applied to the separated substances, which, by changing theseparation conditions, expand the possibilities of chromatography.

Chromatography allows you to solve problems

analytical: separation, identification, determination.

formulation: purification,

separation, concentration.

Chromatogram of a mixture of two substances

The time from the moment of entering the analyzed sample to themoment of registering the maximum chromatographic peak is calledthe retention time and is designated – tR.The retention time consists of two components:1) the residence time of substances in the mobile phase – tm,2) time of stay in the stationary phase – ts.3) The tm value is actually equal to the time of passage through the

chromatograph of the unsorbable component.The tR value does not depend on the amount of sample entered inthe column, but depends on the nature of the substance and thesorbent, as well as on the packaging of the sorbent, and may varyfrom column to column.Therefore, to characterize the true retention capacity of the column,enter the corrected retention time – t'r= tR-tm.

To characterize retention, the concept of retained volume is used-VR (the volume of the mobile phase that must be passed throughthe column at a certain speed in order to elute the substance):

VR = F×tR,

where F is the volume flow rate of the mobile phase (cm3/s) or (ml /min).

Based on the resulting chromatogram of the mixture, you cancalculate the experimental values of chromatographic parameters: theretention factor (capacity) - K, shows how many times longer thesubstance remains in the stationary phase than in the mobile phase,this value should be from 1.5 to 4 K;selectivity coefficient – a, a measure of the relative retention orrelative mobility of two separated substances;resolution-RS, the separation of two adjacent substances, is a measureof the completeness of the separation of two substances. The split isconsidered complete if Rs is equal to or greater than 1.5.

The amount of the test substance in the sample can be estimated byabsolute calibration of the chromatograph using GSO (state standardsamples), followed by the construction of a calibration graph.Or by internal normalization, where it is assumed that the peaks of allpossible components of the mixture are fixed on the chromatogram,and the sum of their areas (S) is equal to 100%.Or by an internal standard method, in which a known amount of thereference compound is introduced into the analyzed sample.

Column

investigated material

Components

Chemical composition

Ion exchange chromatography (IEC) is a method that separates ionsand polar molecules based on their charge.

The interaction of the sample and the sorbent is based on a reversibleCoulomb interaction.

The sorbent has ionic functional groups on the surface that interactwith the analyte ions of the opposite charge.

Cation-exchange IEC – a negatively charged sorbent bindspositively charged molecules of the analyzed substance (analyte).

Ani-exchange IEH-a negatively charged analyte binds a positivelycharged sorbent.

IEC is used to separate almost any charged molecules, includinglarge proteins, small nucleotides, and amino acids.

Ion exchange chromatography

A typical chromatogram of ion exchange separation

Affinity chromatography is the separation of biological moleculesbased on the selective interaction between a protein (or proteincomponent) and a specific ligand associated with the matrix.The method allows you to purify the substance (increase itsconcentration) by several thousand times. Affinity chromatography isa unique purification technology because it is the only one that canpurify biomolecules based on their biological functions or chemicalstructure.

Stages of affinity separation of proteins

Hydrophobic chromatography (HPLC) is based on the hydrophobicity ofproteins. Proteins containing hydrophobic amino acids on the surfacecan reversibly bind to the hydrophobic groups of the sorbent.

A typical chromatogram of the separation of hydrophobic

Gel filtration (GF) or exclusive chromatography (sieving, gel-penetrating, gel-filtration chromatography) is a method of separatingmolecules according to their size.Unlike ion exchange and affinity chromatography, sample molecules donot bind to the sorbent, but the smaller the molecules, the faster they willpass through the gel layer.An important advantage of GF is that the environment conditions can bechanged according to the sample type or requirements for furthercleaning and storage steps.GF is well suited for biomolecules that are sensitive to changes in pH,concentration of metal ions, cofactors, and other environmentalconditions.Separation can be performed in the presence of the necessary ions,cofactors, detergents, urea, guanidine hydrochloride, at high or low ionicstrength, at 37°C or in the cold, according to the requirements of theexperiment.

Gel filtration

Chromatographic system Bio-Rad BioLogic LP

ELECTROPHORESIS

Electrophoresis is the movement of particles of the dispersed phase(colloidal or protein solutions) in a liquid or gaseous medium underthe action of an external electric field.It was first discovered by professors of the Moscow University P. I.Strakhov and F. F. Reiss in 1809.There are two types of electrophoresis:cataphoresis - the surface to be processed has a negative electriccharge (that is, it is connected to the negative contact of the currentsource) andanaphoresis – the charge of the treated surface is positive.

Vertical electrophoresis - allows for high-resolution separation of proteins and DNA. Two-dimensional gel electrophoresis (2D) and isoelectric focusing. Horizontal electrophoresis - allows separation of proteins and DNATGGE gel electrophoresis with temperature gradient. Currently, the main application of gel electrophoresis with a temperature gradient is the screening of mutations and analysis of microbial populations, heteroduplex analysis, the study of DNA methylation, analysis of the secondary structure of RNA, the study of protein-protein interactions and analysis of protein thermal stability. Pulsating field electrophoresis is the ideal solution for separating high-molecular-weight DNA.Immunoelectrophoresis.

Gel electrophoresis is a method of spatial separation of DNA,proteins, or macromolecules in General by their weight (or charge).

Pulse-electrophoresis

Nucleic acid hybridization

Nucleic acid hybridization

Cutting byrestrictionenzymes

Electrophoresis in agarose

gelTransfer to

a filter

ChromosomalDNA

DNA fragments

Incubation of a filter with a radioactive

probeWashing and

exposure of the filter with film

Film development

Position of the fragment

homologous to the radioactive probe

Southern blot hybridization scheme

The sequence of nucleotides that contains the sites recognized bydifferent restriction enzymes (In: Alikhanyan and others, 1985, P. 415)

Partial cutting DNA with restriction enzymes results in the appearance of a series of overlapping fragments with the same sticky ends. Vertical arrows mark restriction sites, and horizontal arrows mark DNA fragments (In: Russel, 1998, p. 459)

Synthetic primer Single-strand DNA hybridization

protein sequence

DNA sequenceDNA sequence

The picture of the separation of oligonucleotides

Sequence of DNA sequencing operations

Gel electrophoresisvisualization of fragmentsreading the sequenceof DNA fragments ACGANG

2 е- + 2Н2О →2ОН- + Н2↑

Cathode (-) Anode (+)

Н2О →2Н+ + 1/2О2↑ + 2 е-

е-

е- е-

е-

е-

е-→

→ →→

→

→

Basic information about the theory of electrophoresis 1. In the solution between the electrodes, the current is due to the bufferand sample ions, and in the rest of the circuit, to the electrons.2. The current in the circuit is maintained by electrolysis occurring onthe electrodes, each of which is immersed in a large buffer chamber.

Sample Electric field Buffer Carrier

1. Charge

2. Sizes

3. Form

4. Weight

1. Amperage

2. Voltage

3. Resistance

1. The composition

of the buffer

2.The concentration

of the buffer

1. Adsorption

2. Electro-osmosis

3. Molecular sieve

Factors affecting the mobility of substances in electrophoresis

Example of an electrophoresis

Negatively charged proteins move towards the cathode. Proteins with asmaller mass move faster. The density of staining of the spots is higher,the more protein it contains. Blurring of spots is caused by the presenceof isoforms of the same type of protein, which differ slightly in aminoacid composition and, accordingly, in mass, but not in function.

buffer solution serum start linesupporting environment (media)

electrophoresis line

start linealbumine

globuline

Determination of protein molecular weight by electrophoresisAliquots of proteins with a known mass (standard) and separately –aliquots of proteins with an unknown mass are entered. Since thespeed of movement of proteins in an external electric field dependson their mass, the mass of an unknown protein is calculated using adirect proportion of the known speed of its movement (distance fromthe start line/duration of electrophoresis) and the speed of movementof standard proteins with a known mass.

Charge. Mobility increases with the total charge. The charge valueusually depends on the pH.

Sizes. The larger the molecules, the less their mobility; this is due toan increase in the friction forces and electrostatic interactions of largemolecules with the environment compared to smaller molecules.

Form. Molecules of the same size but different shapes, such asfibrillar and globular proteins, have different mobility; this is due todifferences in the friction force and electrostatic interaction.

Weight. The larger the mass of the molecule, the less mobile it is.

ELECTRIC FIELD

According to Ohm's law, the current I (in amperes),voltage V (in volts) , and resistance R (in ohms) arerelated by the following ratio: I = V/R. All three factorsaffect the separation of ions in an electric field.

Amperage.Since the current in the solution between the electrodes isdue solely to the transfer of buffer and sample ions, thespeed of their movement is directly proportional to thecurrent strength.The length of the path traversed by the ions will beproportional to the time of current transmission.Therefore, for maximum reproducibility of results, thecurrent strength during the electrolysis process should notchange.It goes without saying that the current must be constant.

Voltage. It is related to the current strength of the aboveratio; it follows that the migration rate is proportional tothe voltage drop in the supporting medium, or the voltagegradient, usually expressed in V/cm-1 (the applied voltagedivided by the length of the carrier layer).Both low (100-500 V) and high (500-10, 000 V) voltages withgradients up to 20 and 200 V/cm-1 are used, respectively.

Resistance. The migration rate is inversely proportional tothe resistance, which in turn depends on the type and sizeof the carrier and on the ionic strength of the buffer.The resistance increases with increasing length of thecarrier layer and decreases with increasing width, as wellas with increasing concentration of buffer ions.

ELECTRIC FIELD

The buffer creates and stabilizes the pH of the carrier, as well asaffects the rate of migration of substances in various ways.

The composition of the buffer. The most widely used buffers areformate, acetate, citrate, veronal, phosphate, Tris, EDTA and pyridine.To separate carbohydrates, borate buffers are often used, the advantageof which is that they form charged complexes with carbohydrates.

The buffer concentration. As the ionic strength of the bufferincreases, the current component due to the transfer of buffer ions willincrease, and the proportion of current due to sample ions willdecrease. Thus, the migration speed of the sample will decrease. Whenthe buffer has a high ionic strength, the total current increases, andtherefore the amount of heat generated increases.At a low ionicstrength, the current due to the transfer of buffer ions decreases, andthe proportion of current due to sample ions increases. Thus, themigration of the sample is accelerated. In a buffer with a low ionicstrength, the total current and heat output decrease, but the diffusionincreases, resulting in a lower resolution than with a high ionicstrength.

CARRIERRelatively inert substances are used as carriers, but theircomposition is still not indifferent to the mobility ofdifferent substances, and the choice of the appropriatemedium therefore depends on the nature of the sample.

Adsorption. Adsorption — retention of sample moleculesby the carrier, as in adsorption chromatography. This leadsto blurring of spots on the chromatogram, as a result ofwhich the sample does not move in a clear band, but has theappearance of a comet; the resolution of the method isreduced. Adsorption also leads to the reduction of speed ofmigration. Paper has the greatest ability to adsorb, but thisundesirable property can be eliminated if you use celluloseacetate.

Electroosmosis (electroendosmosis), this phenomenon iscaused by the appearance of a relative charge between thewater molecules of the buffer solution and the surface of thecarrier. Ionization of carrier groups and surface adsorptionof buffer ions usually leads to the formation of hydroxonium(H30 -) ions from water molecules. Since these ions arepositively charged, they move to the cathode, capturing thedissolved neutral substances and speeding up the movementof cations; the speed of movement of anions decreases.Usually these effects can be ignored, however, if theisoelectric point of a substance is determined, an appropriatecorrection must be introduced. This is usually done bymonitoring the movement of electrically neutral compounds,such as urea or glucose.

CARRIER

Molecular sieve. The properties of a molecular sieve are those of ahard carrier (gel) used in gel electrophoresis.these properties help toseparate mixtures of charged macromolecules, such as proteins, whichdiffer not only in their electrophoretic mobility, but also in their shapeand size. Gels consist of randomly intertwining molecular chainsdistributed throughout the gel volume and forming a sieve-likestructure. According to the specific requirements of separation, thepore size of gels can be varied within certain limits. The principle ofoperation of the molecular sieve in agar, starch and polyacrylamidegels is that large molecules move through it the slower the smaller thepore size, which is determined by the number of cross-sections in thegel. When using sefadex-type gels, the situation is reversed.

CARRIER

The buffer-rich carrier on which the sample is deposited is usuallypositioned horizontally (horizontal electrophoresis) on a flat surfaceof an insulating material, such as plexiglass.

In horizontal electrophoresis, separation can be performed on paper,cellulose acetate, in gel plates, and in a thin layer, although workingwith thin layers at higher voltages requires the use of metal coolingplates, as in high-voltage electrophoresis.

Device for horizontal electrophoresis

The buffer-rich carrier on which the sample is deposited is usuallypositioned vertically (vertical electrophoresis) on the vertical surfaceof the insulating material, such as plexiglass.In vertical electrophoresis, separation is usually carried out in a

polyacrylamide gel.

Device for vertical electrophoresis

BLUE COOMASSIE

Electrophoretic separation of proteins by isoelectric focusing occursunder the action of an electric field in the pH gradient in accordancewith their isoelectric point (PI). The location of each protein isdetermined by the value of its isoelectric point. When a proteinreaches an isoelectric point in the pH gradient, its total chargebecomes zero and it stops moving in the electric field. As a result ofelectrophoretic separation of the test substance, protein moleculescan diffuse from the focus zone, but when they get into a moreacidic or alkaline environment, the protein molecules will lose theirneutrality and return to the zone of the isoelectric point under theinfluence of an electric field.

The pH gradient is formed in the tube, dispersing the electrolyte in an electric field

A solution of proteins is placed and dispersed in an electric field

After staining, individual proteins are visible as stripes

In this method, an electric field is applied to the proteins in thestarch gel first in one direction, and after acceleration – in thesecond direction, perpendicular to the first, which allows for morecomplete separation of the analyzed proteins if there is an overlap ofproteins similar in amino acid composition.

Temperature Gradient Gel Electrophoresis (TGGE) is based on the separation of biomolecules by temperature gradient in accordance with their melting point. When passing through the temperature gradient, the DNA begins to melt. After the chains diverge, a fragment of single-stranded DNA significantly slows down the movement in the gel. Fragments of double-stranded DNA with a higher melting point continue to move in the gel. The sooner the DNA chains separate, the sooner the fragment stops in the gel. Since the melting point of DNA depends on the composition of nucleotides, electrophoresis in the temperature gradient allows you to separate DNA fragments of the same length, but different nucleotide composition.

The system is based on the separation of biomolecules by temperaturegradient in accordance with their melting point.When passing through the temperature gradient, the DNA begins tomelt. After the chains diverge, a fragment of single-stranded DNAsignificantly slows down the movement in the gel. Fragments of double-stranded DNA with a higher melting pointcontinue to move in the gel. The sooner the DNA chains separate, the sooner the fragment will stopin Gelendzhik. Since the melting point of DNA depends on the composition ofnucleotides, electrophoresis in the temperature gradient allows you toseparate DNA fragments of the same length, but different nucleotidecomposition.

A. Perpendicular TGGE: to determine the optimal temperature gradient for sample separation.

B. Parallel TGGE: multiple samples are separated simultaneously. Used for routine separation.

Unlike conventional ROFE devices with fixed electrodes, the systemis equipped with free rotation electrodes. Thus, any angle can be setbetween the periodically changing directions of the electric field. Theaccuracy and high speed of changing angles is achieved due to thedesign features of the rotor. This advantage of the system isparticularly important for separating very large DNA molecules.

Immunoelectrophoresis — IEF) is a method for studying the antigenic composition of biological materials that combines electrophoresis and immunodiffusion. First described by Grabar and Williams in 1953, in 1965 the method was modified by Scheidegger to minimize (the so-called micromodification of the IEF method). A sample of antigen material is separated by electrophoresis in a gel (usually agarose), resulting in the formation of characteristic zones. Further, a precipitating antiserum is introduced parallel to the electrophoresis zones, the antigen and antiserum diffuse towards each other, and at the meeting point of the antiserum with the antigen, precipitation lines appear in the form of an arc. After immunodiffusion and elution of non-precipitated molecules from the gel, the gel is colored with special dyes.

ElectrophoresisIn PAAG (polyacrylamide gel) and starch gel 16-18 fractions

immunoelectrophoresis - about 30 fractions

Immunoelectrophoresis of human blood proteins (for Henry)

Albuminα1-Lipoproteineα1-Glicoproteineα1-Antytripsinα2-MacroglobulineGaptoglobinePre-β-lipoproteineTrasferrinβ-lipoproteineComplementIgAIgMIgGPrealbumin

Capillary electrophoresis

The method of capillary electrophoresis is based on the separation ofcomponents of a complex mixture in a quartz capillary under theaction of an applied electric field. The micro-volume of the analyzedsolution is introduced into a capillary pre – filled with a suitablebuffer-electrolyte. After applying a high voltage (up to 30 kV) to theends of the capillary, the components of the mixture begin to movealong the capillary at different speeds, depending primarily on thecharge and mass (more precisely, the value of the ion radius) and,accordingly, at different times reach the detection zone.The resultingsequence of peaks is called the electrophoretogram, wherein thequalitative characteristic of a substance is the retention parameter(migration time) and quantitative – height or peak area is proportionalto the concentration of the substance.

BLOTTING

BLOTTING

Blotting – a set of techniques that include the transfer of separatedmacromolecules from a certain medium (for example, gel) to a carrier(special paper, nitrocellulose filters, etc.).There are two main types of blotting – capillary (for example,southern blotting), which is based on the movement of molecules dueto the capillary effect, and electroblotting, in which the transfer ofmolecules is provided by electrophoresis.

Western blotting-transfer of separated proteins from the gel

to the carrier

Southern blotting-transfer of specific nucleotide sequences of DNA from a gel to a carrier

Northern blotting-transfer of specific RNA nucleotide

sequences from a gel to a carrier

Eastern blotting - the process of detecting posttranslational

modifications of a protein

Blotting-transfer of separated macromolecules from a certain medium (for example, gel) to a

carrier

Southern - blotting-detection of specific nucleotide sequences bytransferring electrophoretically separated DNA fragments fromagarose gel to a nitrocellulose (paper) filter due to the capillary effect(blotting – " blotting») and hybridization with a labeled DNA or RNAprobe that is complementary to the desired sequence. The formation ofhybrids is detected by autoradiography. The method was developedBy E. Southern and R. Davis in 1975.

Northern blotting is a method similar to the southern blotting methodused for testing RNA fragments and molecules (instead ofnitrocellulose, a filter made of diazobenzyloxymethyl cellulose isused, and complementary DNA molecules are used as probes. TheNorthern blotting method was proposed by J. Bolvan and hiscolleagues in 1977.

BLOTTING

Eastern blotting-detection of posttranslational modifications of proteins.

Western blotting (protein immunoblot, Western blotting) is an analytical method used to determine specific proteins in a sample. Western blotting was developed in the laboratory of George Stark at Stanford. The name Western blot was given to the technique by W. Neal Burnett (W. Neal Burnette) and is a play on words from the name southern blotting, a technique for determining DNA developed earlier by Edwin Southern.

BLOTTING

The blot-hybridization method (Southern-blot) (E. Southern, 1975)

Northern blotting

1. Separation of proteins by SDS-PAGE gel electrophoresis/ Using gel electrophoresis, the proteins are separated in a polyacrylamide gel. 2. Transfer of proteins onto membrane.3. Blocking and Detection.Then they are detected using antibodies: first, the proteins bind to primary (mono - or polyclonal) antibodies, which in turn bind with secondary antibodies conjugated to enzymes (horseradish peroxidase or alkaline phosphatase).

Protocol

Western blotting can detect antigen in amounts less than 1ng

4. Visualization.A high degree of resolution is

achieved due to the electrophoretic separation of proteins and the specificity of monoclonal antibodies.

Visualization of the studied protein is achieved by conducting an appropriate biochemical reaction with the formation of a product that is determined by colorimetric, chemiluminescent, and fluorescent detection methods.

5. Analisis

The amount of protein is estimated using densitometry.

Southern Blotting This method identifies unique fragments of DNA, the size of which is approximately one millionth of the genome

Genomic DNA (usually isolated from white blood cells or fetal cells) is split into short fragments, separated in agarose gel, transferred to the membrane, and then identified specific areas by hybridization with oligonucleotide probes.

Nothern Blotting

Analog of Southern Blotting.This method allows you to identify a specific mRNA and estimate its size.

Eastern Blotting (it is a continuation of the Western blotting method)

Defining the Western Blotting method

The method is based on a combination of gel electrophoresis and immunochemical reaction "antigen-antibody".

"Solid phase" for immunoblot

• porous materials such as nitrocellulose (PVDF) in the form of fillers in volume or in the form of flat sheets or strips of strips;

• strips are used in methods such as immunoblot and immunochromatography; in porous materials, the area where one of the interaction participants is sorbed is significantly larger;

• other reagents diffuse through the pores.

Types of solid phase for Western blotting

Copper cathode2ē + 2H2O→H2 + 2OH-

Upper buffer gelThe

cath

ode

plat

e

Polyacrylamide gel with samples

MembraneBottom buffer gel

Copper anodeCu → Cu2+ + 2ē

The

anod

e pl

ate

Sample preparation• The sample can be taken from whole tissue or from cell culture. In most cases, solid tissue is first ground mechanically using a blender (for large volume samples), using a homogenizer (smaller volumes), or ultrasound treatment. • Different detergents detergents, salts and buffers can be used to improve the lysis of the cells and solubilize proteins. • Protease and phosphatase inhibitors are often added to prevent samples from being broken down by their own enzymes. • Tissue preparation is often performed at low temperatures to avoid protein denaturation.

One-piece tissue Cell culture

Mechanical grinding

Grinding with a homogenizer

Ultrasound treatment

Grinding in liquid nitrogen

Detergents, salts, buffers

Protease and phosphatase inhibitors

Con

ditio

ns t

hat

impr

ove

ssam

ple

prep

arat

ion

Low temperature

produces homogenization of the samples due to their shaking the microtubes or vials with solid balls

Gel electrophoresis. The most common method of

protein separation is electrophoresis in

polyacrylamide gel in the presence of Lammi SDS

Gel electrophoresis

• SDS causes denaturation of proteins and maintains them in a denatured state, reducing agents of disulfide bonds are used to destroy secondary and tertiary structures of proteins

Gel electrophoresis

The proteins to be analyzed in the presence of sodium dodecyl sulfate acquire the same negative charge, which makes it possible to separate them depending only on the molecular weight.

Principle of electrophoresis

Pre-denatured proteins are brought into the pockets of" tracks " (tracks) of an acrylamide gel with a low concentration (concentrating gel), which allows them to be concentrated before passing into a separating gel (with a higher concentration), where the proteins are separated depending on the molecular weight. Proteins migrate in an electric field through the acrylamide gel to the anode, while smaller proteins move faster.

Principle of electrophoresis

Differences in the speed of advance-electrophoretic mobility leads to the separation of proteins into strips.

As a rule, one of the "tracks" is left for markers of molecular weight (a mixture of proteins with known masses).

Staining of gels

To visualize the results of electrophoresis most often use the staining of proteins in gels with Coomassie dye or silver

coloring of proteins in gels with Coomassie dye

staining proteins in gels with silver

Analysis of electrophoretic separation of proteins

• In most cases, the results of electrophoretic separation are sufficient to obtain by visual evaluation of the gel.

• However, in order to obtain reliable data and properly document the results, the gel is scanned for lumen using a highly sensitive densitometer, which allows us to reliably determine not only the position of proteins in the gel, but also the optical density of the protein spot.

Membrane staining is more reliable

normal

paraproteinemia

M-gradient in the gamma globulin zone

Albumin

Globulins

Analysis of electrophoretic separation of proteins, Blotting

• Using a special software application, you can determine such parameters as the electrophoretic mobility of the protein, its purity, the amount of protein in the spot, etc.More often, chemiluminescent protein detection system is used – the use of x-ray films (Blotting)

Use a software application ImageJ

Analysis of electrophoretic separation of proteins

• Determining the molecular weight of the test protein requires calibration of the gel by molecular weight. The gel is calibrated relative to the molecular weight of protein markers, which are separated in parallel with the test sample.

Hepar Brine Hepar Brine

Select % resolving gel

• the concentration of acrylamide determines the resolution of the gel — the higher the concentration of acrylamide, the better the separation of low-molecular-weight proteins. A low concentration of acrylamide improves the resolution of gel electrophoresis for high-molecular proteins.

The protein size, kDa

%AA

36-205 5%

24-205 7.5%

14-205 10%

14-66 12.5%

10-45 15%

Transfer to the membraneTo make the proteins available for antibodies and further detection, they are

transferred along with a strip of gel to a membrane made of nitrocellulose or PVDF

• The membrane is placed on top of the gel, and a stack of filter paper is placed on top of it. The protein transfer method is called electroblotting and uses an electric current that transfers proteins from the gel to the membrane. Proteins move from the gel to the membrane while maintaining their location. As a result of this "blotting" process, the proteins are held on a thin surface layer of the membrane for detection. Both versions of membranes are used because of their non-specific protein binding properties. Protein binding is based on both hydrophobic interactions and electrostatic interactions between the membrane and the protein. Nitrocellulose membrane is cheaper than PVDF, but much more brittle and less resistant to repeated tagging.

filter papergelnitrocellulosefilter paper

Types of electroblotting

• Dry• Wet• Semidry

Confirmation of protein transfer to the filter (Ponceus color)

Blocking• Once the membrane is selected,

the antibodies and the target protein are selected, measures must be taken to exclude interaction between the membrane and the antibody used to detect the target protein (for the antibody itself is a protein). Blocking non-specific bindings is achieved by placing the membrane in a dilute protein solution-usually bovine serum albumin or low-fat milk powder or gelatin with a small percentage of the detergent type Tween-20.

Blocking is one of the most important stages of effective Western blotting

The blocking mechanismThe protein from the dilute solution attaches to the membrane in all places where the target protein has not attached. Therefore, when adding antibodies, they (antibodies) have no free space on the membrane where they can attach, except for binding sites on specific target proteins. This background "noise" in the final Western blot product leads to clean results and the elimination of false positives.

Detection. The next step is the binding reaction of the test protein with a specific antibody (primary)

• The antibody solution and the membrane can be closed together and incubated for 30 minutes before being left overnight. They can also be incubated at different temperatures, and better binding is observed at elevated temperatures.After removing the unbound primary antibodies, the membrane is maintained with secondary antibodies and according to their target properties, usually called by the type "anti-mouse", "anti-goat".

Antibodies for Western blotting. The mechanism of detection

• Antibodies are obtained from an animal source and bind to most primary antibodies. Secondary antibodies are usually bound by alkaline phosphatase or horseradish peroxidase. The most common horseradish peroxidase-related secondary antibodies are used to cut the chemiluminescent agent, and the reaction product produces luminescent radiation in proportion to the amount of protein.

A sheet of photosensitive photographic film is placed against the membrane and exposed to the reaction radiation, creating an image of antibody bands on the blot. A cheaper, but less sensitive approach using 4-chlorine naphthol staining in a mixture with 1 % hydrogen peroxide, which gives a dark brown staining that is registered without the use of a special photographic film.

Detection. Other detection methods

Another method of secondary antibody detection uses antibodies with a bound fluorophore that emits in the near-infrared (NIR) region. The light emitted by the fluorescent dye is constant and makes fluorescent detection a more accurate and sensitive way to measure the difference in the signal produced by antibody-labeled proteins on the Western blot.

The third alternative method uses a radioactive label instead of an enzyme linked to a secondary antibody (with a radioactive isotope of iodine). Other methods are safer, faster, and cheaper, so radioactive detection is rarely used.

Detection. Other detection methods

Visualization

Visualization is performed using gel-documenting systems or a digital camera.

Polymerase chain reaction (PCR)

PCR method – this is an effective way to increase the number of specific DNA

sequences millions of times over a short period of time

99

100

101

What is needed for PCR?

• A specific DNA sequence (target DNA) with a length from 100 BP to 35 BP.;

• Two synthetic oligonucleotide primers (priming) flanking the target DNA and oriented in the direction of 5’-3' in its direction.

• After annealing, the primers hybridize with opposite DNA strands, with their 3’ - hydroxyl ends oriented towards each other;

• Thermally stable DNA polymerase (Taq polymerase); • Four dNTP

Annealing with primers55-67 °С

Denaturation93-95 °С

Each stage is repeated

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Standard PCR is performed automatically in a thermal cycler

• The change in the reaction type is set by changing the temperature of the

reaction mixture: • 95 (0.5 min)• 55 (1.5 min)• 72°С (I min)

PCR is performed in a single tube containing:

about 1 mcg of DNA, 20 picomoles of each primer, 50 micromoles for each dNTP; two units of thermally stable Taq DNA

polymerase.

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How is the effect of multiplying a specific DNA sequence achieved?

By repeatedly repeating three consecutive reactions:

Denaturation of the target DNA The reaction mixture containing the above components is kept at a temperature

of 95°C;

Renaturation The temperature of the mixture is slowly lowered to 55 °C, and the primers bind to the complementary

sequences of the target DNA; DNA synthesis The temperature is raised to 72° C,

and the synthesis of complementary DNA chains begins,initiated by 3’ - hydroxyl groups of primers.

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How real-time PCR works?PCR real time

• Real-time PCR uses a TaqMan system that controls PCR kinetics directly during amplification using resonant fluorescence quenching.

• For detection, a probe is used that is complementary to the middle part of the amplified fragment.

• The probe carries a fluorophore and a fluorescence extinguisher, When the fluorophore and quenching agent are connected to an oligonucleotide probe, only minor fluorescent emission is observed.

• During PCR, the DNA probe is attached to the complementary DNA chain during the primer annealing stage,

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PCR real time

• During the elongation stage, Taq polymerase synthesizes a complementary DNA chain and when reaching the probe, it begins to cleave the probe due to the presence of 5'-exonuclease activity.

• In this case, the separation of the fluorescent label and the extinguisher occurs, which leads to an increase in the detected glow.

• It is obvious that the more amplicons have been accumulated during PCR at a given time, the more intense the glow will be

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Multiplex Real-Time PCRIn modern versions of real-time

PCR, several fluorescent probes labeled with different fluorescent dyes are used simultaneously.

This allows multiple PCR products to be detected simultaneously in a single test tube.

Many modern "real-time" PCR devices provide options for detecting multiple fluorescent dyes simultaneously. Detection of the fluorescent signal from each dye occurs in a certain range for it.

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Nobel prize in chemistry 1993

Received Kary B. Mullis

For the development of the polymerase chain

reaction method

Секвенирование – установление последовательности аминокислот в белке, либо нуклеотидов в нуклеиновых кислотах.

Реакция Сэнгера – не ПЦР, а многократный синтез с одной матрицы

Complementary interactionsSanger sequencing

1 before Sanger sequencing, the DNA molecule is cut into fragments and cloned in Escherichia coli. Fragments isolated from bacterial cells are repeatedly amplified using a polymerase chain reaction (PCR).

2 PCR consists of the following. The DNA sample is heated to a temperature at which there is a divergence of the chains. Then deoxynucleoside triphosphates (dNTP) and a primer — a short oligonucleotide complementary to a small segment of the DNA matrix-are added to the reaction mixture. It hybridizes with this segment, and DNA polymerase sequentially attaches to its end dNTP, complementary to the nucleotides of the copied chain. The process is repeated many times until millions of copies of each fragment are obtained.

3 the Solution with single-stranded fragments and primers is distributed in four test tubes, each containing four different dNTP and one of the fluorescently labeled dideoxynucleoside triphosphates (ddNTP). Elongation of the primer hybridized with the DNA fragment occurs until ddNTP is included in the chain. At this point, the synthesis stops, and as a result, a unique set of negatively charged fragments of different lengths is formed in each of the test tubes, ending in one of the labeled ddNTP.

4 The fragments are separated by size using capillary electrophoresis. When fragments of a certain length pass through the detector window illuminated by a laser beam, ddNTP begin to fluoresce. The length of the fluorescence wave depends on which ddNTP is at their end, so the output is a color image that can be transformed into a nucleotide sequence.

CHAIN EXTENSIONA single-stranded fragment of DNA, called a matrix, together with a short oligonucleotide complementary to its terminal section, is fixed on the substrate (a). Add fluorescently labeled deoxynucleosidetriphosphates (dNTP) and a polymerase that attaches to the end of the primer a dNTP complementary to the corresponding matrix link (b). The unbound dNTP and polymerase are removed using a laser, excite the fluorescence of the nucleotide attached to the primer and identify it (C). Remove the fluorophore and continue to lengthen the chain.

CHAIN EXTENSION

LIGATIONA single-stranded matrix is attached to a primer adjacent to the segment of the matrix DNA that they want to sequence. Short oligonucleotide probes are Synthesized, which contain one of the four nucleotides at a given position — A, T, G, or C.After one of the probes finds a complementary nucleotide in the matrix, the ligase stitches it with the primer. This moment is recorded, the probe is identified, the complex is removed from the matrix, and the entire procedure is repeated.