2 replies to this topic

[gyma]

Hi I just got a problem in getting the whole cell lysate of several T cell lines. Here is my protocol:
1. collect cells and wash with PBS once.
2. add RIPA buffer and 4C rotate for 30 min
3. 15000rpm, 4C for 20 min
4. store the supernatant at -80C.

RIPA:
50mM Tris-Cl, pH8
150mM NaCl
2mM EDTA
1% Triton X-100
0.1% SDS

I add protease inhibitor cocktails in PBS and RIPA before use.

As you may know, T cells have very big nuclei and quite limited cytoplasm. The problem is after rotation at 4C for 30 min, for some T cell lines, all nuclei were disrupted. I have checked that by microscopy. But for the others, I can see big debris (either viscous or not) by nake eye and many remaining nuclei by microscopy. I tried sonication and that did get rid of the debris for me. However, the IB result didnot look good with the sonicated samples. So I really want a protocol to completely lyze the cells without sonication or other extra procedures. My target protein localizes in both cytosol and nucleus and I want to compare the expression level between these cell lines.
Thank you very much.

Edited by gyma, 25 July 2011 - 05:42 AM.

[chabraha]

run them through a 20G needle/syringe several times or treat them with DNAse/MNase to make soluble the nuclear material.....or even better increase your salt concentration to 250mM......this will extract salt sensitive chromatin binding proteins. Also with sonication you have to make sure that you are not over-sonicating your sample........this leads to heating and foaming of the sample which in turn ruins your proteins......very low settings with 10-20 brief 1sec pulses should be more than enough.

[gyma]

chabraha, on 28 July 2011 - 08:21 AM, said:

run them through a 20G needle/syringe several times or treat them with DNAse/MNase to make soluble the nuclear material.....or even better increase your salt concentration to 250mM......this will extract salt sensitive chromatin binding proteins. Also with sonication you have to make sure that you are not over-sonicating your sample........this leads to heating and foaming of the sample which in turn ruins your proteins......very low settings with 10-20 brief 1sec pulses should be more than enough.

thank you very much. I will try 250mM NaCl.
I have used 30G, 26G needles in order to reduce the viscosity. For 30G, I couldnt even suck up the lysate. For 26G, I did but all lysate turned into foam finally. I just dont think that could sufficiently break the nuclei.
For sonication, I used a setting as 30s on, 30s off. for some samples, I need 10 mins, for others maybe 20 mins or more to completely get the viscosity away.