Having trouble detecting BRCA2 protein by Western Blot.
I used a 3-8% Tris-Acetate gel, 40 ug of protein and ran the gel for 4 hours. To transfer I ran the wet apparatus at 4C overnight at 25 volts. Still there was no detection. Would a reducing agent such as BME help?
Hi Rachel,
There are a couple of things to check.
BRACA1 is located in the nucleus, is your lysis buffer/method harsh enough to break the nuclear membrane. Our lab had some issues earlier with this where freeze thaw wasn't sufficient with out buffer and sonication was required. If you pellet out the nucleus when you spin down your sample, you won't get your protein.
3-8% are very low percentage gels and it shouldn't be difficult to transfer off a protein that is 250KDa. I run 4-16% gels for a 180KDa protein and I transfer at 350mA for 40min and all of the protein has left the gel (including the 250KDa ladder).
I think you should try and stain the gel after transfer to see if you are getting complete transfer (coomassie), and also stain the membrane to see that you can see it on the membrane (ponceau) before running the rest of the western blot. This should tell you if your protein is in the gel/on the membrane.
For your particular experiment, why are you not using BME in your loading dye? Generally it is quite inmportant to ensure a nice blot-unless of course you are doing native gels where protein interactions are important to preserve.
Good luck,
Jacob
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