I see a double band for PSD-95. Wondering what it could be. It seems to be there due to an artifact (not enough beta-Mercapto, maybe) it does not show up in samples of another batch. The additional band shows up right above the regular band. Slightly longer exposure merges the bands, this is how close they run. I only see the additional band in one group of brain lysates (mutants), not wildtype (or its much milder). I wonder if it could be Palmitoylation? Would I be able to see such a shift in a regular blot with regular lysates (given the idea the beta-ME was not reducing sufficiently/at all)? Neddylation/Ubiquitination? Who has experience with PSD-95 double bands? THanks for any advise!
Lipid anchor. Your protein is membrane-associated, and I think by lipid anchors. Check if the anchor is removable; that would mean that you're seeing two population of this protein - with the anchor and without the anchor, on WB it would look like two very close bands ("double band").
The CST anibody datasheet shows that phosphorylated PSD95 could be shifted (shift lost by phosphatase treatment).
https://www.cellsignal.com/products/primary-antibodies/phospho-psd95-ser295-a8f8z-rabbit-mab/45737
Phosphorylations can be labile and non-reproducible to detect if the lysates are not handled properly. Add enough phosphatase inhibitors in your lysis buffer or directly lyse samples in 2x loading dye, boil for 5-10 min and load to retain most modifications. You can also test the above antibody to see whether your knockout could be affecting phosphorylation at this site (obviously, other phosphosites could also be involved in the shift).
Best, Manoj
Thanks Yulia and Manoj.
I also thought about palmitoylation. But I was not sure how easy it is to see by accident. Since the protocols I see are rather complicated. And Westernblots to show palmitoylation with a shift are not so easy to find. I foun done for PSD95, but as I said, sample preperation is complicated. Phosphorylation I kind of exclude in this case. The samples where I see the shift have no phosphatase inhibitors. Other samples that have phosphatase inhibitors show one band, only.
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