SDS-PAGE Gel Electrophoresis

SDS-PAGE Gel Electrophoresis Table of Contents

What is SDS-PAGE Gel Electrophoresis?
Background
Applications
SDS
Separation
Migration
Procedure
Stacking Gel
Resolving Gel
Materials
Buffers
Resolving Limits of SDS-PAGE Gels
Preparation
Protocol
Visualization
Gel Analysis
SDS-PAGE How To Videos
SDS-PAGE Videos
Other SDS-PAGE Protocols
Troubleshooting SDS-PAGE Gels
Discussion Topics for Agarose Gel Electrophoresis
Get Help on your Agarose Gel Problem
Tips for Successful SDS-PAGE Gels

Definition of SDS-PAGE?

Definition: SDS-PAGE is an abbreviation for sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis.

What is SDS-PAGE?

SDS-PAGE is a molecular biology technique used to separate proteins accordingly by size. SDS-PAGE also can separate DNA and RNA molecules.

In what is probably the most powerful technique for resolving protein mixtures, proteins are exposed to ionic detergent SDS (sodium dodecylsulfate) before and during gel electrophoresis. SDS denatures proteins, causing multimric proteins to dissociate into their subunits, and all polypeptide chains are forced into extended conformations with similar charge:mass ratios. SDS treatment therefore eliminates the effects of differences in shape so that chain length, which reflects mass, is the sole determinant of the migration rate of proteins in SDS- polyacrylamide electrophoresis.

Even chains that differ in molecular weight by less than 10 percent can be separated by this technique. Moreover, the molecular weight of a protein can be determined by comparison to a protein ladder or molecular weight ladder which is run on the same gel.

(see Figure 1)
sds-page gel

Figure 1. This acrylamide gel is used to separate proteins. The colored spots are standard-size markers, which are prestained. This is usually used to make a western blot. The proteins are transfered to a membrane and then detected with an antibody to a specific protein.

Role of SDS in SDS-PAGE Gel Electrophoresis

SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis, is a technique used in biochemistry, genetics and molecular biology to separate proteins according to their electrophoretic mobility (a function of length of polypeptide chain or molecular weight as well as higher order protein folding, posttranslational modifications and other factors).

The solution of proteins to be analyzed is first mixed with SDS, an anionic detergent which denatures secondary and non–disulfide–linked tertiary structures, and applies a negative charge to each protein in proportion to its mass. Without SDS, different proteins with similar molecular weights would migrate differently due to differences in mass charge ratio, as each protein has an isoelectric point and molecular weight particular to its primary structure. This is known as Native PAGE. Adding SDS solves this problem, as it binds to and unfolds the protein, giving a near uniform negative charge along the length of the polypeptide.

SDS bind in a ratio of approximately 1.4 g SDS per 1.0 g protein (although binding ratios can vary from 1.1-2.2 g SDS/g protein), giving an approximately uniform mass:charge ratio for most proteins, so that the distance of migration through the gel can be assumed to be directly related to only the size of the protein. A tracking dye may be added to the protein solution to allow the experimenter to track the progress of the protein solution through the gel during the electrophoretic run.

protein gel electrophoresis

Electrophoresis Seperates Molecules according to their Charge:Mass Ratio

Electrophoresis is a technique for separating, or resolving molecules in a mixture under the influence of an applied electric field, also called electrophoretic mobility.. Dissolved molecules in an electric field move, or migrate at a speed determined by their charge:mass ratio. For example if two have the same mass and shape, the one with the greater net charge will move faster toward an electrode. The separation of small molecules, such as amino acids and nucleotides, is one of the many uses of electrophoresis. In this case, a small drop of sample is deposited on a strip of filter paper or other porous substrate, which is soaked with a conducting solution. When an electric field is applied as the ends of the strip, small molecules dissolved in the conducting solution move along the strip at a rate corresponding to their magnitude of their charge.

SDS-Polyacrylamide Gel Electrophoresis Pore Size

Because many proteins or nucleic acids that differ in size and shape have nearly identical charge:mass ratios, electrophoresis of these macromolecules in solution results in little or no separation of molecules of different lengths. However, successful separation of proteins and nucleic acids can be accomplished by electrophoresis in various gels (semisolid suspensions in water) rather than in a liquid solution. Electrophoretic separation of proteins is most commonly performed in polyacrylamide gels. These gels are cast between a pair of glass plates by polymerizing a solution of acrylamide monomers into polyacrylamidechains and simultaneously cross-linking the chains into a semisolid matrix. The pore size of a gel can be varied by adjusting the concentration of polyacrylamide and the crosslinking reagent.

When a mixture of proteins is applied to a gel and an electric current applied, smaller proteins migrate faster than larger proteins through the gel. The rate of movement is influenced by the gel’s pore size and the strength of the electric field. The pores in a highly cross-linked polyacrylamide gel are quite small. Such a gel could resolve small proteins and peptides, but large proteins would not be able to move through it.

SDS-PAGE Procedure

As mentioned, the proteins are mixed with SDS an anionic detergent which binds to the proteins and denatures their secondary and non–disulfide–linked tertiary structure with the addition of heat. SDS also applies a uniform negative electrical charge to each protein in proportion to its mass.

If SDS was not used in separation, proteins with similar molecular weights would migrate differently in the gel due to differences in their mass to charge ratios. As proteins are separated using electrical current and pore size of the gel matrix, differences in charges (in addition to mass) would play a role in separation.

Tracking Dye for SDS-PAGE

A tracking dye may be added to the protein solution to allow the experimenter to track the progress of the protein solution through the gel during the electrophoretic run.

SDS-PAGE Protocols

SDS-PAGE Electrophoresis Protocols

Protein Electrophoresis Protocols

SDS PAGE Protocols

sds-page gel

electrophoresis

sds-page

running a gel

sds-page analysis

SDS-PAGE Gel Electrophoresis Forum Topics

protein quantification Hi, can any one let me know how to quantify proteins secretes by a fungus extracellularly/ thank you.........
Transferring, except that it hasn't transferred! Hi there, I'm having a problem with transferring. Basically I set everything up, wet transfer, beautiful. Transferred for 100mins at 250mA (same...
Air bubbles in PAGE Hi all, I have problems with air bubbles between gel and the glass plate while casting PAGE. I am not able to figure out the reason for...
SDS gradient gel problems Hi all, recently I ordered some gradient gels from BioRad, and I noticed that when I am running samples the dye smears and forms weird shape (like an...
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SDS-PAGE Electrophoresis Videos

Demystifying SDS-Page Video

An instructional video aimed at teaching the process of SDS-PAGE to college students with basic biochemical understandings. www.sdspage.net From Youtube user mattbrewbaker.

Added by: kiki06
Tags: sds-page gel electrophoresis
Date: 2008-04-28

SDS-PAGE Gel Electrophoresis Video