Team:Cambridge/Protocols/Gel Electrophoresis of Protein

Protein Analysis by SDS PAGE

These are some notes made in preparation for running SDS PAGE to verify we have Reflectin.

A 12% PAGE gel will separate 12kDa-60kDa proteins

Theory

SDS-PAGE or sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis is a method of resolving proteins of different molecular weights (kDa) by mixing samples with SDS, loading samples into usually an acrylamide gel and passing an electric current through it.

SDS is 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, allowing fractionation of proteins via electrophoresis similar to a DNA gel with longer proteins experiencing more difficulty moving through the gel than shorter proteins. Samples are often heated in boiling water prior to loading to shake up the molecules and allow improved binding with SDS. A tracking dye for example bromophenol blue is used to indicate the stopping point.

After electrophoresis the gel is rinsed in D.I water and stained with a dye commonly coomassie blue, PAGEBlue or silver staining for improving fainter bands for visualisation of the separated proteins. After staining the gel is rinsed again and left to de-stain to your desired amount either in D.I water or in a de-staining solution.

There are protocols to make gels of the desired percentage weight of SDS and there are also pre-cast gels available from Bio-Rad and Life Technologies. We ran our gels using pre-cast 12% gels sourced from Bio-Rad using Bio-Rad electrophoresis tanks.

Practice

How to do it in the lab

{Standard layout for procedures is to use:

• <Procedure title - aka what you are doing>

1. <step 1>
2. <step 2>
   • <additional notes/important information regarding the previous step>

the text within the < > is what should be written, don't include < > in actual writeup :P

if in doubt see the gel electrophoresis protocol

}

Safety

The safety implication of the procedure.