Hi dear everyone,
I had a problem in extracting total protein from T cells. I used a RIPA buffer which is composed by
50 mM Tris-Cl pH8
100 mM NaCl
I used this buffer for IP in 293FT cells before and it worked well. Also some labmate used this buffer for IB in T cells and it worked well. But in my case, I couldnt lyse the nucleus with this buffer in T cells. I had observed the lysate under microscopy and the nuclear membrane remained intact. I even lysed it overnight and still didnt work. Needles helped but since I have lots of samples so I just want a good buffer that could give me total cell lysate without any additional work.
To solve this problem, I will try to make a new batch of this buffer and see if something wrong happened to this old one. Also I did a lot of search and found almost all RIPA buffers (either commercial or not) contain sodium deoxycholate (half of those contain 0.1% SDS). Since SDS seems to be able to break the nuclear membrane, so I will try to make another RIPA with SDS. But I dont have sodium deoxycholate, I am wondering if it is ok without it? Thank you very much.
RIPA buffer without sodium deoxycholate
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