Running a western with cell media in gels? - (Jan/13/2011 )
mdfenko on Wed Jan 19 17:31:28 2011 said:
cm13 on Wed Jan 19 16:39:20 2011 said:
Protein is about 90kda.
Think the main serum proteins are at 70? is that right?
which is close enough to distort your protein if overloaded.
At present im thinking i should centrifuge the media sample (at what speeds anyone?)
And then use 20-30ul of the pellet in an SDS gel.
And then use 20-30ul of the pellet in an SDS gel.
there is a calculation for "clearing time" of a protein by ultracentrifugation (it may work in a superspeed but may take a long time). it is dependent on the sedimentation coefficient of your protein and the rcf at which you spin (i can give you the calculation, if you really want it, it's in the beckman ultracentrifuge log book). you could try to pellet the protein, blindly, by spinning at >100 000xg for a few hours.
If i do a coomassie blue stain, would that show my protein, or would i have to go ahead and run the western (transfer + antibody incubation, etc) ?
yes, you can stain the gel (with coomassie or silver, depending on the protein load). you should be able to make out the band of your protein if it is in sufficient quantity and not obscured by the serum proteins.
I talked with my supervisor - He said it's that im trying to get the microparticles from the cell media and investigate if the protein is presnet in them. (Endothelial cell media. cant remember if i mentioned that)
So i've to pre-spin the media, and then spin it at 20000g at 4C for i think 10 minutes
And then i'll run the pellet on a western under normal conditions.
Does this seem right? Whats the best way to extract these microparticles?
-cm13-
yes, it should work. suspend in loading buffer and heat.
-mdfenko-