My protein is embedded in the inner membrane and does not run clearly on gel, but rather appears smeared at the top of the lane since it probably links other debris. Also, the epitope appears to be masked as a result.
I want to free it some more, but without using ultracentrifugation.
Hi Sylvie,
you write "smear on a gel at the top of the lane". Question, what kind of gel? SDS/polyacryleamide? Could you lower the percentage of acrylamide so that your protein gets better into the gel? I also found out that some protein need a higher concentration of mercaptoethanol or ditiothreitol in the loading buffer to run properly on SDS gels.
Good luck!
Yes, it is SDS page. The protein is about 46 kDa, but on Western instead of seeing a clear band I see smudges high up on the lane, indicative that the protein is attached to components that hinder migration.
Disrupt the cells by sonication or other physical method. Do a low-speed spin (5,000 g for 10-20 minutes) to pellet large fragments and unbroken cells. Then spin the supernatant at the highest speed you can achieve with your standard centrifuge (a benchtop microcentrifuge, for example, can usually achieve 16,000 g or more) at 4oC. Spin for as long as you can, because the longer you spin, the better the yield of total membranes. Also, using small tubes is helpful because the shorter the liquid column, the faster the membranes reach the pellet. Then, extract the membrane pellet with 1% N-lauroylsarcosine (Sarkosyl), which dissolves the inner membranes, leaving the outer membranes intact. Centrifuge again for as long as you can to pellet the outer membranes. The supernatant is your inner membrane extract.
- Badges
- Science method