Hi there,
I am using cold methanol fixation (1 volume cold methanol to 1 volume cell culture medium) and then lyse my cell. Since method damages the cell membrane am wonder whether my protein of interest could be (partly) be dissolved in the methanol/medium solution. For this I would like to do a western blot.
I am concerned that the high amount of methanol in my sample disturbs the SDS-Page. Is it sufficient to check that the pH is above 4.6 or do I first need to somehow remove the methanol from my sample?
- If it were me I would wash my fixed material with cold PBS before lysing for western blotting
Hi,
I would consider adding some protease inhibitors and speed-vac'ing the sample. Additionally, you can use TCA to precipitate all proteins and dissolve it in loading buffer.
Good luck!
Using a speed-vac as proposed by Anton Slyvka is probably the easiest solution. If your sample contains 50% methanol, it will probably freeze in the speed vac very quickly. Therefore, I am not so sure protease inhibitors are necessary. You would then have a dry pellet, which you can take up directly in sample buffer.
Stop the speed-vac, when a tiny bit of frozen liquid is still with the sample. This helps dissolving them. Completely dry samples sometimes do not dissolve very well.
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