FAQ
How are protein samples concentrated?
There are a number of methods that can be used for concentrating protein samples that are too dilute for immediate electrophoretic analysis. These include TCA precipitation, ammonium sulfate precipitation, lyophilization and dialysis. Samples concentrated by any of the latter three methods should be dialyzed against 62.5 mM Tris-HCl (pH 6.8) for use in the Laemmli SDS discontinuous buffer system. Dialysis removes the salts which interfere with electrophoresis. (For example, if not removed, potassium ions will precipitate SDS. Also, salts in general cause localized increases in current, which can lead to uneven migration rates across the gel.)
Concentration by TCA Precipitation
1. Precipitate the protein by addition of 10% TCA. Allow to incubate on ice for 30 min.
2. Centrifuge for 5 min at 12,000 x g.
3. Gently and repeatedly wash the pellet with ethanol-ether (1:1 v/v) to remove TCA.
4. For Laemmli SDS-PAGE, TCA precipitates are dissolved directly in 62.5 mM Tris-HCl (pH 6.8), 2% SDS, 5% 2-mercaptoethanol, 10% sucrose or glycerol, 0.002% Bromophenol Blue. The dye color should remain blue. If there is TCA still present, the dye will be yellow. The pH must then be adjusted by adding µl volumes of concentrated Tris base until the dye becomes blue/purple. (Note: Excess TCA must be neutralized or it will interfere with electrophoresis.)
Reference:
Gel Electrophoresis of Proteins: A Practical Approach, Second Edition, pp 45-46, ed. B.D. Hames and D. Rickwood. Oxford University Press, 1990.