it will be very helpful in learning 2D gel electrophoresis

1. 2D GEL ELECTROPHRESIS
ANN MARY MATHEW
M.Sc. 1st YEAR
BGS051705
11-03-2018

2. WHAT IS SDS-PAGE?
WHATIS THE PRINCIPLE OF SDS-PAGE?
HOW WE SEPARATE TWO PROTEINS WITH SAME
MOLECULAR WEIGHT ?

3. Two-dimensional electrophoresis (2-D electrophoresis) is a powerful and
widely used method for the analysis of complex protein mixtures extracted
from cells, tissues, or other biological samples.
Based on two independent properties :
The second-dimension step, SDS-
polyacrylamide gel electrophoresis
(SDS-PAGE), separates proteins
according to their molecular weights
(Mr, relative molecular weight).
the first-dimension step, isoelectric
focusing (IEF), separates proteins
according to their isoelectric points
(pI);

4. WHAT IS IEF ? WHY IEF?
pH<pI pH=pI pH>pI

5. EXPERIMENTAL PROCESS :
SAMPLE PREPARATION
IPG STRIP REHYDRATION
IEF
IPG STRIP EQUILIBRATIION
SDS-PAGE
VISUALIZATION
ANALYSIS

7. 1. SAMPLE PREPARATION
• Must break all non-covalent protein-protein, protein-
DNA, proteinlipid interactions, disrupt S-S bonds
• Must prevent proteolysis, accidental
phosphorylation, oxidation, cleavage, deamidation
• Must remove substances that might interfere with
separation process such as salts, polar detergents
(SDS), lipids, polysaccharides, nucleic acids
• Must try to keep proteins soluble during both phases
of electrophoresis process

8. CELL DISRUPTION
Gentle Lysis Method – Osmotic lysis, Freeze thaw lysis, Detergent lysis,
Enzymatic lysis.
Vigorous Lysis Method – Sonication, Grinding, Mechanical
homogenization,
PROTEASE INHIBITORS
-PMSF(phenyl methylsulfonyl fluride), AEBSF(aminoethyl benzylsuonyl
fluride),1mM EDTA etc..
PRECIPITATION
- Ammonium sulfate, TCA, acetone, TCA in acetone etc..
CONTAMINANT REMOVAL
– Filtration, Centrifugation, Chromatography, Solvent Extraction

9. PROTEIN SOLUBILIZATION
– 8 M Urea (neutral chaotrope)
– 4% CHAPS (zwitterionic detergent)
– 2-20 mM Tris base (for buffering)
– 5-20 mM DTT (to reduce disulfides)
– Carrier ampholytes or IPG buffer (up to 2% v/v) to enhance
protein solubility and reduce charge-charge interactions
- 0.002% bromophenol blue

10. 1ST DIMENSION : ISOELECTRIC FOCUSSING

11. IEF & IPG (IMMOBILIZED pH GRADIENT)
•Separation on the basis of pI, not MW
•Available in different pH ranges
3-10
4-8
5-7
•Requires very high voltage
Acrylamide monomer
R- weakly acidic or basic
buffering group

12. Amersham Biosciences Bio-Rad
1-D Instruments……

16. Or….
Place the IPG strip on the IEF apparatus and apply current

17. 2nd Dimension – SDS PAGE
equilibration

18. Casting IPG on 2nd Dimension

19. Visualization……
Desired features……..
• High sensitivity
• Wide linear range for quantification
• Compatibility with mass spectrometry
• Low toxicity and environmentally safe
• Environmentally friendly

20. Autoradiography and fluorography are the most sensitive detection
methods. To employ these techniques, the sample must consist of
protein radiolabelled in vivo using either 35S, 14C, 3H or, in the case
of phosphoproteins, 32P.
For autoradiographic detection, the gel is simply dried and exposed
to X-ray film or—for quicker results and superior dynamic range of
quantification—to a storage phosphor screen.
Fluorography is a technique that provides extra sensitivity by
impregnating the gel in a scintillant such as PPO (2,4-
diphenyloxazole) prior to drying.

24. Further Analysis of Protein Spots
• Picking the spots
Ettan Spot picker is a robotic system that automatically picks
selected protein spots from stained or destained gels using a
pick list from the image analysis, and transfers them into
microplates.
• Digestion of the proteins
supernatant peptides are mixed with MALDI matrix material
and spotted onto MALDI slides using Ettan Spotter.
• MALDI-ToF mass spectrometry
In the Ettan MALDI-ToF mass spectrometer, a laser beam is
fired into the dried peptide-matrix spots for ionization of the
peptides. De novo sequencing of proteins.