Sharpen Up Your Protein Gels
Last week, Nick talked about about SDS-PAGE. Today I am going to tell you about tweak that will improve your SDS-PAGE protein gels.
It involves using Bis-Tris gel buffers. Although Bis-Tris adds a considerable cost to the technique, it has several advantages:
1. Bis-Tris gels are acidic, in contrast to the alkaline conditions found in conventional SDS-PAGE gels. This supresses cytosine reoxidation, which prevents proteins from cross-linking via di-sulphide bonds in the gel.
2. Sodium bissulphate, a reducing agent, is present throughout the buffer system. Unlike conventional PAGE, this means that the reducing environment is maintained all the way through the gel, which also helps to prevent disulphide bond formation.
3. Low molecular weight proteins do not run faster towards the end of the gel.
4. Conventional PAGE gels degrade after a month or two as the acrylamide breaks down to acryclic acid. This does not happen with Bis-Tris gels, which means they have a much longer shelf life.
Together, these factors mean that the resolution obtained on Bis-Tris SDS-PAGE gels is significantly greater than with conventional SDS-PAGE gels.
Here’s how to make a Bis-Tris gel:
5X low-MW running buffer Used for separating small proteins 2-50 kDa.
250 mM MES
250 mM Tris
5 mM EDTA
0.5% SDS
Sodium bisulfite to 5 mM (add fresh before run) from a 1M stock.
5X high-MW running buffer use for separating proteins >20 kDa.
250 mM MOPS
250 mM Tris
5 mM EDTA
0.5% SDS. add sodium bisulfite to 5 mM (add fresh before run) from a 1M stock.
200X running buffer reducing agent
1 M sodium bisulfite add to running buffer at 5 mM final concentration
3.5X gel buffer 1.25 M bis-Tris (pH 6.5-6.8 with HCl)
Note: bis-Tris is Bis(2-hydroxyethyl) aminotris (hydroxymethyl) methane (e.g. Sigma catalog# B 7535).
The method is not very different from the conventional methods of casting protein gels, just replace the tris buffer that you use in the stacking and resolving gels with the Bis tris buffer and omit SDS from the gel. Run at at a constant voltage of 150V.
Bis-tris gels were developed by Tim Updyke and Sheldon Engelhorn for Invitrogen. Similar gels are marketed by Invitrogen under the NuPAGE label.
If you try this, or already use them, let us know how these gels work for you.
Openwetware article on Bis-Tris gels
About the Author
Bala Chandar
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