2-D gel Electrophoresis of proteinn.pptx

Protein separation method
2 D GEL ELECTROPHORESIS IS IMP METHOD

1. 2-D GEL ELECTROPHORESIS BY- PRERANA SAHU 1 st Year Msc Ag (MBB) Adm. No.-231221212

2. WHAT IS 2-D GEL ELECTROPHORESIS? Two dimensional gel electrophoresis (2DE) is a technique used to separate proteins based on their size (sodium dodecyl sulfate -polyacrylamide gel electrophoresis, SDS-PAGE) and their charge (isoelectric focusing, IEF) in the presence of an electric field. 2-DE was first independently introduced by O'Farrell and klose 1975  

3. PRINCIPLE:- In 2D GE proteins are separated as per isoelectric point and protein mass. Separation of the proteins by isoelectric point is called isoelectric focus (IEF). When a gradient of pH is applied to a gel and an electric potential is applied across the gel, making one end more positive than the other. At all pH values other than their isoelectric point, proteins will be charged. If they are positively charged, they will be pulled towards the negative end of the gel and if they are negatively charged they will be pulled to the positive end of the gel. The proteins applied in the first dimension will move along the gel and will accumulate at their isoelectric point; that is, the point at which the overall charge on the protein is 0 (a neutral charge). In separating the proteins by molecular weight, the gel treated with sodium dodecyl sulfate (SDS) along with other reagents ( SDS-PAGE in 1-D). This denatures the proteins (that is, it unfolds them into long, straight molecules) and binds a number of SDS molecules roughly proportional to the protein’s length, Since the SDS molecules are negatively charged, the result of this is that all of the proteins will have approximately the same negative charge ratio as each other.

4. PRINCIPLE CONTINUE….. In addition, proteins will not migrate when they have no charge (a result of the isoelectric focusing step) therefore the coating of the protein in SDS (negatively charged) allows migration of the proteins in the second dimension. In the second dimension, an electric potential is again applied. The proteins will be attracted to the more positive side of the gel (because SDS is negatively charged) proportionally to their mass-to-charge ratio. The gel therefore acts like a molecular sieve when the current is applied, separating the proteins on the basis of their molecular weight with larger proteins being retained higher in the gel and smaller proteins being able to pass through the sieve and reach lower regions of the gel.

5. PROCESS:- Sample preparation Isoelectric focusing (first dimension) SDS-PAGE (second dimension) Visualization of proteins spots Identification of protein spots

6. Sample preparation:- Must break all non-covalent protein-protein, protein-DNA, protein-lipid 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 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 Protease inhibitors PMSF(Phenyl Methane Sulfonyl Flouride ), Pefabloc , EDTA,, leupeptin, Aproteinin , Pepstatin Contaminant removal Filtration, Centrifugation, Chromatography, Solvent Extraction

7. 1D: IEF (Iso-electric focusing)and IPG(Immobilized pH gradient) :- Separation of amphoteric substance based on charge (reacts as acid as well as base) When pI = pH at which proteins carries no net charge. Acidic pH - Proteins Positively Charge Basic pH- Proteins Negatively Charge IPG strips are the strips of paper made by covalently integrating acrylamide and variable pH ampholytes. Available in pH ranges 3-10, 4-8, 5-7 and requires very high voltages (5000V) and long period of time (10h). Ampholytes are molecules that contain both acidic and basic groups Proteins will be introduced into strips and placed inside the IEF Chamber and electric field will be passed through it then the protein will migrate in the matrix and will find their pH equilibrium( pI ).

8. PLACIN G IPG STRIPS IN IEF CHAMBER SEPARATION OF PROTEINS IN IPG STRIPS

9. 2D:SDS–PAGE (Sodium Dodecyl Sulphate PolyAcrylamide Gel Electrophoresis ) Separation of protein based on Molecular weight Role of 2-Mercaptoethanol and SDS

10. Buffer and reagent for electrophoresis 1. Tetra Methyl Ethylene Diamine (TEMED) 2. Ammonium persulfate (APS) 3. Tris-HCl 4. Glycine 5. Bromophenol blue 6. Coomassie brilliant blue R250 7. Sodium dodecyl sulphate 8. Acrylamide 9. Bis-acrylamide 10. 2-Mercaptoethanol 11. Butanol \ Isopropanol

11. Preparation of Resolving Gel

12. Preparation of Stacking Gel

13. STACKING & RESOLVING GELS STACKING GEL 4% of acrylamide Ordering/arranging and conc. the macromolecule High pore size pH 6.8 RESOLVING GEL 15% of acrylamide The actual zone of separation Less pore size pH 8.8 Stacking Gel Resolving gel

14. Buffer Glycin exist In different form Low pH (+ve) Neutral pH High pH(-ve) Glycin

15. When electric gradient is passed through gel , proteins are separated according to their molecular weight and here no two proteins having same molecular weight overlap each other at same spot due to their different isoelectric point which solves the problem overlapping proteins in 1D SDS PAGE electrophoresis.

16. Detection Of Proteins 1. Using chemical stains like Silver Nitrate Coomassie Brilliant Blue Western Blot 2. 2D gel analysis software Bio Numerics 2D, Delta2D, Image Master PD Quest, Progenesis

17. ADVANTAGES:- Identifies protein modifications Separates proteins by size and charge Allows protein quantification Can analyze many proteins simultaneously Useful for comparing different samples

18. DISADVANTAGES:- Time-consuming process Limited protein detection Difficulty in reproducibility Complex data analysis Inefficient for membrane proteins

19. APPLICATIONS:- Whole proteome analysis Detection of biomarkers and disease markers Cancer research Cell differentiation Bacterial pathogenesis Drug discovery Microscale protein purification Product characterization Comparative proteomics: identifying and analyzing differences between complex mixtures of proteins Protein profiling, biomarker discovery Separation and analysis of protein variants and isoforms