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2 d gel electrophoresis

This document summarizes a seminar presentation on 2D electrophoresis. 2D electrophoresis is a technique used to separate mixed proteins based on their isoelectric point and mass. It involves two sequential electrophoretic steps: iso-electric focusing to separate proteins by charge, followed by SDS-PAGE to separate by molecular weight. The document describes the principles, methods, applications and references for 2D electrophoresis.

Science◦
DR. AMBEDKER COLLEGE,DEEKSHABHOOMI, NAGPUR DEPARTMENT OF BIOTECHNOLOGY AND BIOCHEMISTRY SEMINAR ON 2D-Electrophoresis Presented by PIYUSH GHOSHE M.Sc. Biotechnology Semester-1st
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.
Based on two independent properties 1st Dimension Iso-electric Focusing (pI) pH at which a protein has a neutral charge • Separation based on Charge. • Material used - Acrylamide + ampholytes +Riboflavin. 2nd Dimension SDS-PAGE • Separation based on molecular weight. • Material used -SDS Buffer.
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
• 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.
Chemicals used during IEF • Ampholytes • Riboflavin • Acrylamide • Spacer ( insulation tape)
Preparation of gel Ampholytes, Riboflavin are mixed with Acrylamide solution Pour it over Glass plate containing Spacer and cover it by 2nd glass plate Photopolymerization Occur under Bright Sunlight Decomposition of Riboflavin (formation of free Radicals) Polymerization Occurs (require 2-3 hrs) and Glass plates are removed
2D: SDS-PAGE (Sodium Dodecyl Sulphate PolyAcrylamide Gel Electrophoresis) Role of 2-Mercaptoethanol and SDS • Separation of protein based on Molecular weight
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
Preparation of Resolving Gel Acrylamide solution 12% (acrylamide & bisacrylamide) Mixed with TEMED and APS Add Tris HCl and Poured in between the glass plate fitted into the gel cassette Allow them to Solidify and A thin layer of organic solvent i.e. butanol or isopropanol Is Added
Preparation of Stacking Gel Acrylamide solution 4% (acrylamide & bis-acrylamide) Mixed with TEMED and APS Add Tris HCl and Poured in between the glass plate Above the Resolving gel. Allow them to Solidify Then insert a IEF strip over it
STACKING & RESOLVING GELS STACKING GEL • 4% of acrylamide • Ordering/arrangin g 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
Glycin exist In different form Low pH (+ve) Neutral pH High pH(-ve) Glycin Buffer cathode Anode
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
Applications • Analyze proteins • Separation of proteins • Determination of molecular mass • To determine amino acid sequence • Separation of DNA
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/Garfi n_IEF_webArticle9-07.pdf • http://en.m.wikipedia.org/wiki/Isoelectric_point • http://m.youtube.com/watch?v=YV4gR4vucwY