SDS-PAGE

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SDS PAGE :

SDS PAGE Submitted by k.parvathi bai pt/2010/15

Stacking gel:

Stacking gel The stacking gel is a large pore polyacrylamide gel (4%). This gel is prepared with Tris buffer pH 6.8 of about 2 pH units lower than that of electrophoresis buffer. These conditions provide an environment for Kohlrausch reactions, as a result, proteins are concentrated to several fold and a thin starting zone of the order of 19 ?m is achieved in a few minutes. This gel is cast over the resolving gel. The height of the stacking gel region was always maintained more than double the height and the volume of the sample to be applied.

Resolving gel:

Resolving gel The resolving gel is a small pore polyacrylamide gel (3 30%). The Tris buffer used is of pH 8.8. In this gel, macro molecules separate according to their size. In the present experiment, 8%, 10% and 12% Resolving gel were used for separating different range of proteins. 8% gel for 24 – 205 kD proteins, 10% gel for 14-205 kD proteins and 12% gel for 14-66 kD proteins

SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE):

SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE) SDS (Sodium Dodecyl Sulfate) detergent solubilizes and denatures proteins negative charge to proteins Heat denatures proteins

principle:

principle Electrophoresis is the study of the movement of charged molecules in an electric field. The generally used support medium is cellulose or thin gels made up of either polyacrylamide or agarose. Cellulose is used as support medium for low molecular weight biochemicals such as amino acid and carbohydrates whereas agarose and polyacrylamide gels are widely used for larger molecules like proteins.

poly-acrylamide gel electrophoresis:

poly-acrylamide gel electrophoresis PAGE is based upon the principle that a charged molecule will migrate in an electric field toward an electrode of opposite sign . There are two types of PAGE experiments: • SDS-PAGE (99.9% of applications) • Native PAGE (0.1% of applications)

Seperation of proteins:

Seperation of proteins

CONTENTS :

CONTENTS Principle Procedure protocol applications Methods of devolopment

What is so special about SDS?:

What is so special about SDS? SDS is a negatively charged detergent. Disrupts secondary and tertiary protein structures by breaking hydrogen bonds and unfolding protein. ‘Masks’ charge on protein so that all proteins act the same as regards charge. Prevents protein aggregation. Prevents protein shape from influencing gel run

Steps in SDS-PAGE:

Steps in SDS-PAGE Extract Protein Solubilize and Denature Protein Separate Proteins on a gel Stain proteins (visualization) Analyze and interpret results

Uses of SDS-PAGE:

Uses of SDS-PAGE Determine protein size Identify protein Determine sample purity Identify existence of disulfide bonds Quantify amounts of protein Recycle back to the PM. Recycle back to TGN

procedure:

procedure 1. Assembling the glass plate 1. Assemble the glass plate on a clean surface. Lay the longer glass plate down first, then place 2 spacers of equal thickness along the rectangular plate. Next place the shorter glass plate on top of the spacers so that the bottom ends of the spacers and glass plates are aligned (Figure 1).

Uses of sds page:

Uses of sds page Determine purity of proteins. 0. Molecular weight determination NOT dependent on shape {Stokes Radii} as is case for SEC. Western Transfer always begins with SDS-PAGE.

Slide 18:

Antibodies can be produced from proteins excised from gel. Sometimes can reconstitute proteins. Mass spectrometry and proteomic studies.