complete detailed principle of SDS-PAGE components method application mechanism of separation

1. Presenter Dr Anurag Yadav
Post-graduate, biochemistry
Father Muller Medical college
Dr Anurag yadav,Bio-FMMC1
ELECTROPHORESIS

2. 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.

3. 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

4. Dr Anurag yadav,Bio-FMMC4
 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.

5. Components
Dr Anurag yadav,Bio-FMMC5

6. Dr Anurag yadav,Bio-FMMC6
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.

7. Dr Anurag yadav,Bio-FMMC7

8. Movement of
particle
Dr Anurag yadav,Bio-FMMC8
[Cl] > [protein-SDS] >
[Glycinate]

9. Dr Anurag yadav,Bio-FMMC9

10. Dr Anurag yadav,Bio-FMMC10
 In separating gel, protein separate owing to
molecular sieving properties.
 Smaller proteins pass more easily, larger one
retarded by friction.

11. - 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:

12. 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

13. STAINING:
Fluorescent stains - Ethidium bromide – Nucleic
acids
Silver stain for protein gel (sensitive 50 times dye
based)
Dye based – Coomassie blue – 50ng protein
band
Tracking dyes – BPB> xylene cyanol, Orange G

14. Dr Anurag yadav,Bio-FMMC14

15. References
Dr Anurag yadav,Bio-FMMC15
 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.