HELP! acidic SDS-PAGE sample - Has anyone dealing with this before? (Jul/31/2006 )
I've got some acidic protein. They are aggregates that are soluble in 70% formic acid. This sample appear as a horrible smear in SDS PAGE. (I'm using 5% stacking and 10% resolving with pH 8.8)
If I increase pH of the sample before adding lysis buffer, the protein would precipitate out and become insoluble. Since it cannot dissove in SDS.
How could I produce a beautiful PAGE in this case?
If it is not very solube in the running buffer you would expect to see a smear. If it is an acidic protein, raising pH would help its solubility, rather reduce it. If it is also hydrophobic, then SDS would help. I dont have a good answer with so limited info provided.
Thanks for your suggestion
I would like to clearify: the protein is not acidic, but it is soluble in acid. Therefore while I denature it with 1:1 (v/v) 2x SDS-PAGE sample buffer (pH 6.8), the protein somehow precipitate out.
The running buffer I use is the standard SDS running buffer, and pH of stacking and resolving gel is 6.8 and 8.8 respectively (just following procedure of molecular cloning
)Is there any alternative gel combination available? I have tried to google "acidic SDS-PAGE" but it returns no answer.
check this post for a native acidic page system:
http://www.protocol-online.org/forums/inde...&st=0&#
you may be able to add 0.1% sds to it but i haven't tried.
for the sample, just add sds and 2-me (to the same concentration as the standard sds sample buffer) to the soluble sample.
hope it works
http://www.protocol-online.org/forums/inde...&st=0&#
you may be able to add 0.1% sds to it but i haven't tried.
for the sample, just add sds and 2-me (to the same concentration as the standard sds sample buffer) to the soluble sample.
hope it works
Thanks a lot. I will try it out.
1 more question. How about the acrylamide concentration in stacking and resolving? Is it 5% and 10% respecively as in the denaturing gel system? Also the AP concentration
the stacking gel concentration is normally around 3% (plus or minus) but you can use whatever you feel is appropriate. the same goes for the resolving gel concentration. the original procedures call for 7% but you should use the concentration most suited to your protein.
persulfate should be pretty much the same as you are used to (we use 30ul of 10% for 30ml of gel solution to polymerize in ~1hr, although i don't remember if the acidic environment makes it polymerize differently). just make sure the gel is fully polymerized before using it.