Cell lysing for western blot (Help!) - (Apr/20/2006 )

Hi,

I have being using SDS sample buffer (Tris PH 6.8, SDS 2%) to lyse the cells (all are done at room temperature). (All are done at room temperature). Then boil the samples and determine prot. conc. by BCA. After that, proteins were mixted with DTT, glycerol, and dye before boiling again.

This method works quite well for a number of proteins I detected (such as Beclin 1, Akt, pAkt). I can get one major band on the film. But for one protein called mTOR, there are a lot of bands. The band with correct size is not the strongest one.

Then I begin to consider the possilbe disadvantages of the lysing method I used. Can all proteins includng proteases get denatured under 2% SDS? If you use the similar method to lyse the cells, do u use hot buffer or cold buffer with proteases inhibitors, or just RT buffer (like what I use)?

Thank u very much

You might want to look at other lysis buffer compositions and protocols. I have usually stuck by the current protocol composition with a minor modification here or there with mostly success.

Any suggestions about the lysis buffer? Tks

I ever used RIPA, but got a lot of pellet after centrifugation. For the use of sample buffer, there is barely no pellet (which may mean better protein extraction).

According to the 2nd version of Molecular Cloning, sample buffer at high temp (85 degrees) is used to lyse the cells in dishes. I do not know which one is better, hot buffer or cold buffer with protease inhibitors. I will try these tomorrow.