Hello Dhruti Brahmbhatt

Yes, I have encountered a similar issue and even found a solution to it.

You may face this type of a problem when you use antibodies at a high concentration. You need to titrate your antibodies before you use them for Western Blot.

Secondly, the blocking step is important to prevent non-specific binding. But sometimes the blocking agent may be incompatible with certain antibodies leading to the formation of black patches.

Finally, insufficient washing could be the cause. So, wash your blot with at least 3 changes of wash buffer, for 5 minutes at room temperature for each wash. Also, agitate the blot while washing and don’t limit the amount of wash buffer because the greater the volume of wash buffer used the more efficiently will the blot be washed.

Hope this helps!

Best.

I had an issue with blotches on my Western blots too. Several of us were using the same roll of membrane but not everyone was wearing gloves so it was basically a grungy finger problem.

It can also help to activate the membrane in methanol prior to the transfer step. Our transfer buffer had methanol in it, but it would evaporate (and we made large batches). Basically, you dip the membrane in methanol for about 30 seconds (until the color is even), then proceed with setting up your transfer.

1. Antibody concentration used and blocking time

2. Time to wash and make sure TBST and non-fat milk

3. Time of exposure to ECL and its concentration

Yes, it's likely because of high concentration antibodies, the blocking, or insufficient washing. As for me, I tried to:

1. Reduce antibodies concentration

2. Make a fresh skim milk every time I do blocking (since I use skim milk as blocking agent)

3. Increase the time of washing in every step. At least change the TBST 3 times, for 10 minutes at each.

Hope this helps.