Hi everyone,
I met a problem. I cannot see any signal of brca2. I just made a 4% SDS gel, and did transmembrane for 3 h under 300 mA, but there was no signal when I did ECL exposure.
Because brca2 is such a big protein, 384 kDa, it is a challenge to do WB. However, I didn't find any special protocol for this WB. So does anyone have any experience in BRCA2 WB? or in big molecular weight protein WB?
Please share your methods, THX!!
I'm assuming that you used a positive control of some description? Without loading something that you know contains BRCA2, you essentially have no idea of why it didn't show up on your blot and it could be anything from the protein not transferring to the membrane, to your primary antibody concentration needing to be optimised.
Did you stain the membrane with e.g. ponceau S after transfer to check that the transfer worked correctly? Additionally, stain the gel after transfer with coomassie etc to see how much actually made it out of the gel. By staining both the gel and the membrane you'll have an idea of what happened to the proteins as they may have stayed in the gel, made it onto the membrane or over-transferred and gone straight through onto the pads (a good way to detect over-transfer is to put a second membrane behind the membrane to catch any proteins that pass through, you can then stain the second membrane to check).
Hello Mingqi Li
There are some points you need to consider when performing Western Blot for large molecular size proteins.
1. For large molecular weight proteins I suggest you use Tris-Acetate gel.
2. Large proteins can precipitate out in the presence of methanol. So, you should decrease the methanol percentage (10% or less) in the transfer buffer.
3. Just to ensure that your protein does not precipitate out, you may add SDS to a final concentration of 0.1%. SDS adds uniform negative charge to proteins, making it easier for them to transfer from the gel onto the membrane.
4. For large molecular weight proteins always use PVDF membrane because large proteins can precipitate out in the presence of methanol, and PVDF membrane do not require any methanol in the transfer buffer. So, you have a higher chance of successfully transferring your protein to the blot using PVDF membrane. Also, use PVDF membrane of 0.45um pore size.
5. For large proteins, you should use a wet tank transfer method. As large proteins will transfer out of the gel very slowly, I would suggest you perform overnight transfer at 4°C at 40 mA.
The paper attached below may help you with the protocol. Since you are looking out for one protein, you may use a non-gradient gel.
Article Tris–Acetate Polyacrylamide Gradient Gels for the Simultaneo...
Good Luck!
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