Trying to get protein out of insoluble fraction - (Jan/07/2008 )
After a bunch of immunoblots and immunoprecipitations to characterize my antibody, I found that when I ran an immunoblot on the insolube pellet, and viewed with chemiluminescence, are huge bright band appeared. However, I know my protein is an enzyme that resides in the trans Golgi there are not any articles that suggest that its is associated with the phospholipid membranes.
I am currently taking a 1 million cell pellet, washed to remove serum, and adding 300 ul of RIPA buffer with protease inhibitors and letting in sit on ice for 30 minutes vortexing two are three times during that time, and then store it in the -80C until I use it.
So I thought maybe I should let RIPA work longer before freezing. Also I thought maybe I could use freeze thaw method to really get a good lyse. However, I do not have liquid nitrogen I was thinking I could freeze thaw by simply putting in back in the -80, thaw, and back in -80.
Does anyone have any suggestions.
We use to lyse our cells by mechanical means, ie by passing the cell extracts in a 23 gauge syringe for up to 5 times. This process along with the slightly modified RIPA buffer extract all soluble proteins.
Are there any indications that your protein may interact with insoluble material (keratins, etc)?
What detergent (and at witch concentration) do you add to your buffer?
Are there any indications that your protein may interact with insoluble material (keratins, etc)?
What detergent (and at witch concentration) do you add to your buffer?
To make sure my cell pellet was getting lysed sufficiently, I was using 300ul of extraction buffer consisting of:
0.15M NaCl
0.1 M Tris pH 8
.1% SDS
1% NP-40
with protease inhibitors.
My protein is an enzyme that makes modifications to heparan sulfate glycosaminoglycans (unbranched polysaccharides).
Have you tried Triton X-100 at 1% instead of NP-40?
This detergent is stronger and may help release your protein from the membrane.