I have not been able to see any BRCA2 protein on my blots. I know that this is a high MW protein (390 kDa). Has anyone performed this successfully? Please let me know procedure specifics starting from protein extraction to gel resolution to transfer.
Yes, this is what I followed:
1. Pre-cast gradient gel. 6% might also work.
2. Loaded at least 50 ug total protein; 100 ug is even better.
3. 300mA for 2 hours transfer; 20% methanol for transfer.
4. For preparing the cell lysate, I followed the protocol of Maria Jasin, Cell, 2011.
Good luck !
Are you trying to see protein levels in cultured human cells or tissue? I tried to do WB for BRCA1, which has a similar high MW size. I tried using RIPA buffer for extraction, using 6% SDS-PAGE resolving gel and altering the methanol/SDS concentrations in the transfer buffer to help transfer large MW proteins but I never got publishable results, i.e. the blots were inconsistent and dirty.
My suggestion is to try a high denaturing buffer with 2% SDS/ Tris-based buffer (2x Laemmli's) . RIPA buffer is a mild to low denaturing buffer so you won't get all the proteins associate with nuclear fraction. Also, BRCA2, like BRCA, is most likely in the nuclear fraction so a nuclear extraction may be the way to go. This is easier with cells than tissue. Good luck.
Yes, this is what I followed:
1. Pre-cast gradient gel. 6% might also work.
2. Loaded at least 50 ug total protein; 100 ug is even better.
3. 300mA for 2 hours transfer; 20% methanol for transfer.
4. For preparing the cell lysate, I followed the protocol of Maria Jasin, Cell, 2011.
Good luck !
Dear Rachel,
I must first admit that though I am very much experienced in WB but I never had worked with BRCA. In addition to the helpful comments and suggestions posted by Thomas and Shivsankari I am adding an established protocol for BRCA1 WB freely available from Univ of Michigan.
-Best
Dear Rachel,
I just found a reference for BRCA2 WB where the authors have studied characterization of anti-BRCA2 antibodies in cell lines by Western blot analysis.
Bernard-Gallon D et al-Oncol Rep. 2001 Nov-Dec;8(6):1363-6.
Unfortunately I do not have access to the full text for this article. You might have!
-Best
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