Dr Anurag yadav,Bio-FMMC

Presenter Dr Anurag YadavPost-graduate, biochemistry

Father Muller Medical college

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ELECTROPHORESIS

SDS-PAGE

Dr Anurag yadav,Bio-FMMC2

Sodium dodecyl sulphate- polyacrylamide gel electrophoresis.

Most widely used method for analysing protein mixture qualitatively.

Useful for monitoring protein purification – as separation of protein is based on the size of the particle.

Can also be used for determining the relative molecular mass of a protein.

Dr Anurag yadav,Bio-FMMC3

Mercaptoethanol will break the disulphide bridges.SDS binds strongly to and denatures the protein.

Each protein is fully denatured and open into rod-shape with series of negatively charged SDS molecule on polypeptide chain.

SDS is an anionic

detergent.

The sample is first boiled for 5min in buffer containing • Beta-

Mercaptoethanol

• SDS

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On average, One SDS molecule bind for every two amino acid residue.

Hence original native charge is completely swamped by the negative charge of SDS molecule.

Also referred as Discontinuous gel electrophoresis.

Components

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Glass plates, comb, clamp,Sample buffer, SDS,

Glycerol,Mercaptoet

han--ol, power supply

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Stacking gel: ordering/arranging and conc the macromolecule before entering the field of separation. (4% of acrylamide)• Purpose is to concentrate protein sample in sharp

band before enters main separating gel.Running gel: the actual zone of separation of the particle/molecules based on their mobility. (15% of acrylamide) Pore size: routinely used as 3% to 30% which is of pore size 0.2nm to 0.5nm resp.

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Movement of particle

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[Cl] > [protein-SDS] > [Glycinate]

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In separating gel, protein separate owing to molecular sieving properties.

Smaller proteins pass more easily, larger one retarded by friction.

- Research tool- Measuring molecular weight- Peptide mapping- Protein identification- Determination of sample purity- Identifying disulfide bonds

- Separation of proteins and establishing size- Blotting- Smaller fragments of DNA- Separation of nucleic acids- Major clinical use – ALP separation

APPLICATION:

ADVANTAGES:- Clear, fairly easy to prepare- Exhibit reasonable mechanical strength over

acrylamide conc- Low endosmosis effect

DISADVANTAGES- Gel preparation and casting- exacting n time-

consuming- Complete reproducibility of gel preparation not

possible

STAINING:Fluorescent stains - Ethidium bromide –

Nucleic acidsSilver stain for protein gel (sensitive 50

times dye based) Dye based – Coomassie blue – 50ng protein band

Tracking dyes – BPB> xylene cyanol, Orange G

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THANK YOU

References

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Keith Wilson- Principles and techniques of biochemistry and molecular biology.

Upadhyay- biophysical chemistry.Tietz- Text book of clinical chemistry.Kaplan- clinical chemistry.YouTube and Google images.