I am interested in studying a membrane protein in insect larvae (prohibitin). I'm having trouble visualizing it in western blot.
When the protein extract is stored in freezing, when it thaws, it precipitates. I'm using a lysis buffer 20 mM Tris–HCl [pH 7.5], 300 mM NaCl, 1 mM EDTA, 1% Triton X-100, 2 mM PMSF
I would exchange the Tris against a different buffer compound. Tris shows the worst temperature shift of its pKa with - 0.028delta pKa / delta Kelvin. This means, if you go from 20C to -65C, the pH changes by ~2.4 pH-units.
You can google for dpKa/dK tables and replace it with a buffer that does not change as much. E.g., phosphate has a value 1/10th of Tris.
Since you are interested in doing a Western blot, you can dissolve the sample in the SDS-PAGE sample buffer.
if you what to store the protein add some glycerol to your lysis buffer , and try to not use the EDTA in it.
- 300 mM salt is a lot, 100-150 mM will do and cause fewer problems during electrophoresis.
- A protease inhibitor mix may complement the PMSF.
- Triton is a weak detergent, SDS and/or DOC are stronger if you do not require functional protein.
- Instead of anionic detergents like SDS, you can also use cationic like CTAB, and then do a CTAB-electrophoresis with eastern blotting (doi:10.1016/S0003-2697(02)00639-5). CTAB is milder than SDS and can maintain some proteins in their native conformation (allowing zymograms), yet at the same time it is more solubilising.
- Freeze-thaw cycles are generally bad for proteins, you can avoid them by adding glycerol (eutectic mixture 66.7%, freezing point -46.5 °C) and storage at -20 °C.
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