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.

Like DNA gels PAGE gels can be made with various different weight percentage SDS for different resolutions.

A 12% PAGE gel which we used 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

Before You Begin

Various buffers must be prepared prior to running a gel. If you are making your own gels you will need to make up all the buffers. If you are running a pre-cast gel then you will only require the 4x Sample Buffer, Reservoir Buffer and 4x Upper Tris Buffer. Please note that protocols for making gels vary but they all use the same ingredients.

Reservoir Buffer

4x Upper Tris Buffer

4x Lower Tris Buffer

10% Ammonium Persulfate Solution (TEMED)

4x Sample Buffer

The SDS-PAGE Process

• Gel Making

PAGE gels comprise two parts; a lower resolving gel and an upper stacking gel. The stacking gel is where you load your samples into and in general is twice as tall as the desired wells. The lower resolving gel is where the protein bands will run through and be separated.

Resolving Gel

Stacking Gel

• Preparation of Protein Samples

SDS-PAGE can be run with pure protein samples, mixed protein samples and also directly from cell lysates. The general method for preparing proteins prior to loading is as follows:

1. Check the volume of the wells in the gel. Mix protein samples in volume ratio of 3:1 with 4x Sample Buffer in a microcentrifuge tube.

Note: If you don't have enough samples to completely fill the number of wells dilute the 4x Sample Buffer with D.I water and load this.

1. Submerge the protein in boiling water bath (100^oc works for me) for 2 mins.
2. Centrifuge for 20 secs at 12,000 rpm. You are now ready to load your samples into the gel.

• Running the Gel

• Staining the Gel

• <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.