Chemiluminescent detection

Hi!
So I am new on here and new to working with C-elegans; I will be probing the forum for most of my questions however I did have an initial question. In the chemiluminescent detection stage of my experiment with C elegans, the bands I am specifically looking for are either not showing up or are at a different kda than expected. The last two experiments I applied anti-alpha-synuclein antibody then added anti-actin, and still the band for synuclein showed up after development. This was still the case after stripping the western membrane and adding anti-actin again. Can anyone shed light on their protocol for adding the anti-actin antibody? Also, has anyone had a band show up at a lower or higher kda than expected?

Thanks!
Kaitlyn

I’m not completely clear on what you’re doing. Are you probing the blot simultaneously with two antibodies, or are you stripping and reprobing. If you are stripping and reprobing, are the two primaries raised in different species?

In response to one of your questions, bands can run at different sizes than predicted. Protein secondary sequence and post translational modification can lead to discrepancies in predicted size vs size via western blot. If you’re concerned, you should do an RNAi knockdown experiment to see if you are looking at the correct bands.

best,

Jordan