I had homogenized protein in RIPA buffer but was facing problem in running SDS-PAGE gel. So I precipitated protein with 100% TCA , washed with Acetone and then tried to resolubilize protein in 2X Lammelli buffer. The protein precipitate has become very hard and is not getting resolubilize on heating also. Can anyone suggest the best method to resolubilize the protein pellet as it is not solubilizing now on heating also. Basically I would like to do western blot later.
I suggest that you precipitate the proteins with either cold absolute ethanol or cold acetone. Your problem will be taken care of. What happens in TCA precipitation is that the protein is first getting denatured before precipitation and hence it is not getting resolubilized.
Had this problem before.
1) Try not to percipitete your protein. When you percipitate it, you get huge bits of protein that cannot be processed. Just homogenize your brain, add RIPA, shake at 4C for 2 hours, spin down, get the supernatant, and then solubilize it in Laemmli.
if this doesn`t work:
2) I guess this might be a brain-specific thing, as brain contains lots of fats and other crap. I would recommend you the extended regimen - basically, mash your tissue and perform all the steps mentioned in 1). Then, add DTT + UREA coctail to reduce and denaturate your protein for 30 minutes, and after that you will need to alkylate your proteins with iodoacetamide (this is standard reduction&alkylation protocol - google it). After this is done (takes an hour) you can go ahead with SDS-PAGE. Do not percipitate your protein! Just load a bit of your reducted&alkylated protein mixed with Laemmli buffer and you`ll get awsome bands!
well, I have already precipitated protein in TCA and need a method to resolubilize it now.
- Badges
- Science method