Anti-Bub1 抗体 [2F9] (ab54893)

Thank you for your reply.

You should make sure that your samples are reduced and denatured, meaning with ß-mercaptoethanol or DTT and SDS. For the lysis buffer, since this is a nuclear protein, you will need a strong lysis buffer like RIPA. Or, better yet, you could do a nuclear fractionation. Protocol here: https://www.abcam.com/index.html?pageconfig=resource&rid=11408 Make sure to boil for 10 mins too. Full protocol here: https://www.abcam.com/ps/pdf/protocols/WB-beginner.pdf

RIPA buffer
150 mM sodium chloride
1.0% NP-40 or Triton X-100
0.5% sodium deoxycholate
0.1% SDS (sodium dodecyl sulphate)
50 mM Tris, pH 8.0

You should also decrease the amount of protein you're loading, since 50 ug is quite high and can lead to background. Try loading 20 ug if you're doing a whole cell lysate instead.

Also, a 10% gel may not be the best for such a high MW protein. We'd recommend a 6 or 8% gel for better resolution at higher MW.

Large proteins will tend to precipitate in the gel, hindering transfer. Adding SDS to a final concentration of 0.1% in the transfer buffer will discourage this. Methanol tends to remove SDS from proteins, so reducing the methanol percentage to 10% or less will also guard against precipitation. Lowering methanol in the transfer buffer also promotes swelling of the gel, allowing large proteins to transfer more easily. But since you're using PVDF, methanol can be removed from the transfer buffer altogether, and is only needed to activate the PVDF before assembling the gel/membrane sandwich. Plus, choose wet transfer overnight at 4oC instead of semi-dry transfer.

You should also try switching your blocking buffer to 5% BSA instead of milk. Block for 1 hr at RT or even overnight at 4C. Also include the 5% BSA block in with the primary antibody incubation. You can dilute the primary more to 1:1500 and the secondary to 1:5000 as well.

I hope these suggestions help to improve your results. Since this antibody was purchased almost a year ago, it is outside our Abpromise guarantee. However, we do think that protocol optimization may help in this case.