Dear research gate members,
I am trying to detect all the members of the BCL-2 protein family in T-cell extract. However, normal procedure in proteomics experiments did not work efficiently. Therefore, I thought it would be better to fractionate and isolate these proteins by performing SDS/page electrophoresis on cell lysate.
As I understand that these are thermal stable proteins (5mins heating at 95C in SDS sample buffer is a harsh condition already), but I still a bit skeptical if that would denature all of them. Or should I add Urea 6M to the sample as well? I am afraid of heating Urea wouldn't be such a good idea.
So, if anyone have some experience isolating these proteins, please help and share. Thank you and very much appreciate it.
Hi ,
Bcl-2 family proteins tend to associate with lipids from the mitochondria and that maybe the source of your difficulty. I would consult Douglas Green's and David W. Andrews research papers on this. Both groups have done in vitro and liposome / lipid based interaction type of biochemistry/ biophysics studies, maybe a good start. Good luck,
The remark that Bcl2 family members are mitochondrial membrane associated is correct and some detergents will be necessary to extract them. For proteomics analysis, the extraction followed by the appropriated trypsin cleavage etc and then Mass Spec should work fine. For such a proteomics procedure the SDS extraction would be the most complete way to achieve the recovery of the protein. If you are not looking for protein protein interactions but simply recovery of the Bcl2 then SDS with denaturation would be fine. It sounds like you want to see all members of the Bcl2 family and then doing MS on the entire range of proteins from the cells rather than isolate a particular molecular weight range on a PAGE for elution and MS proteomics would be needed. However since Bcl2 family members would fall in the range of say 15 to 45 kD approximately, you could cut out that MW range from the gel and subject the eluted material to digestion and proteomics. I am sure there are other approaches too.
So if the goal is simply proteomics on Bcl2 family members SDS is best. If the goal is protein protein interactions then a non-denaturing detergent should be used.
I do agree with Dr. Oyler. I have another suggestion. As almost all bcl-2 family proteins have BH3 domain, you can try immunoprecipitation by using specific antibody against BH3 domain followed by gel electrophoresis (preferably 2D gel) and mass-spectrometric analysis. In this case also you may face solubility problem, but you may overcome this by using mild detergents.
Thank you so much Indra, Dr. Oyler, and Dr. Tapas. I actually am performing the same procedure as Dr.Oyler described but I cut the gel from 10kDa to 40kDa. I will have the results within this week. Then maybe I can evaluate and see if I should try Immunorecipitation like Dr. Tapas mentioned. However, with my experience in protein isolation and purification, the SDS page and in gel digestion approaches work really good. I asked the questions to see if there are other better approaches and particularly when it comes to BCL-2 proteins family, what should be taken into consideration here? Thanks.
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