2 d gel electrophoresis

In 2D Gel Electrophoresis proteins are separated as per isoelectric point(pI) and protein mass.

1. DR. AMBEDKER COLLEGE,DEEKSHABHOOMI, NAGPUR DEPARTMENT OF BIOTECHNOLOGY AND BIOCHEMISTRY SEMINAR ON 2D- Electrophoresis Presented by PIYUSH GHOSHE M.Sc. Biotechnology Semester-1 st

2. Introduction Introduction by O'Farrell and Klose in 1975 Separation of mixed proteins Principle In 2D Gel Electrophoresis proteins are separated as per isoelectric point(pI) and protein mass. It is an Combination of Iso-electric Focusing (pI) and SDS-PAGE.

3. Based on two independent properties 1 st Dimension Iso-electric Focusing (pI) pH at which a protein has a neutral charge Separation based on Charge. Material used -Acrylamide + ampholytes +Riboflavin. 2 nd Dimension SDS-PAGE Separation based on molecular weight . Material used -SDS Buffer.

4. 1D: Iso-electric Focusing (pI) 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 Acidic pH Neutral pH Basic pH

5. IEF Gels are made up of Ampholytes-complex mixture synthetic polyamino-polycarboxylic acid. 1-2 mm thick IEF gel Expensive 0.5mm thin layer IEF Ampholytes + Riboflavin in Acrylamide is used. Separation is achieved by applying a potential difference across a gel that contain a pH gradient.

6. Chemicals used during IEF Ampholytes Riboflavin Acrylamide Spacer ( insulation tape)

7. Preparation of gel

8. 2D: SDS-PAGE (Sodium Dodecyl Sulphate PolyAcrylamide Gel Electrophoresis) Role of 2-Mercaptoethanol and SDS Separation of protein based on Molecular weight

9. Buffer and reagent for electrophoresis 1. TetraMethylEthyleneDiamine (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

10. Preparation of Resolving Gel

11. Preparation of Stacking Gel

12. 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 Resolving Gel Stacking Gel

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

14. Detection Of Proteins 1. Using chemical stains like Silver Nitrate Coomassie Brilliant Blue Western Blot 2. 2D gel analysis software BioNumerics 2D, Delta2D, ImageMaster PDQuest, Progenesis

15. Applications Analyze proteins Separation of proteins Determination of molecular mass To determine amino acid sequence Separation of DNA

16. REFERENCE O'Farrell, PH (1975); High resolution two-dimensional electrophoresis of proteins; The Journal of Biologocal Chemistry;Vol.250 NO.10; issue of May 25; 4007–4021pg NPTEL – Biotechnology – Bioanalytical Techniques and Bioinformatics (Module 4 Electrophoretic techniques) 2012,5-9pg Edited by Sameh Magdeldin;Gel Electrophoresis – Principles and Basics; Published by InTech Janeza Trdine 9, 51000 Rijeka, Croatia; 2012;62-65pg http://www.uvm.edu/~vgn/outreach/documents/Garfin_IEF_webArticle9-07.pdf http://en.m.wikipedia.org/wiki/Isoelectric_point http:// m.youtube.com/watch?v=YV4gR4vucwY