Do you know why my membrane looks like this?
Do you think the membrane need wash longer or more times?
I did this western for Lamin B1, and I used ERG sc-376293 from Santa Cruz as primary antibody in 1:1000 and Anti-mouse IgG HRP linked antibody #7076S from Cell Signaling as secondary antiody in 1:2000
I would re-evaluate your blocking solution. If you are using bovine serum albumin (1%) and block for an hour or more, you'll usually find good blocking. Powdered milk is less effective and can give blotchy appearance. You might also consider the membrane itself. We use the LICOR blocking solution and dilute it with 2 volumes of water to make it economical. It works great for our scans.
Besides the issues with blocking solution, the reason may be in the unequal distribution of ECD substrate solution on the membrane.
It also seems to me, that the blot is overexposed. Secondary antibodies from another manufacturer may improve the sutuation, providing more bright signal (that will require less exposition and, thus, less unspecific dots and time for substrate solution to distribute unequally).
Hello Marian
According to my experience, your membrane is not suitable blcoked. I advice you try blocking with non-fat milk in TBS buffer, the incubation period that I use is 3 hours. Blocking with non-fat milk is cleaner than BSA blocking. However, dependent on manufacturer's instructions of your antibody. In addition, in your picture, the spots on your membrane appear to be caused by some impuritues in the western blot trasfer or from buffer. Check it this.
Best regards
Hi
I agree with other researchers. Usually those tiny spots comes from the secondary antibody, it's like it does not conjugate well and then is not properly blocked and washed, so it is like over exposed.
I used to block my membranes with BSA but lately I have been using milk and it works fine for me, you just need to be careful to wash everything really well (TBST 1X) and gentle washing (give it a good shake), to remove unespecific binding and avoid those spots.
Also your bands does not seem really clear to me, I think there was a problem on the transference, but before focusing on problems on the transfer I would dig into the blocking or the washings.
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