Hi, are there other methods beside Western Blotting to detect a specific protein? I‘ve isolated mitochondria and want to show that hPar17/Pin4 is present in mitochondria. The Western Blots didn’t work well, because of the low amount of sample or maybe low expression levels of hPar17 (the Abs work). I want to detect the protein without overexpressing it if possible. I isolate the mitochondria from human cardiac myocytes. Do you have any ideas on how to show the presence/expression of my protein in mitochondria? Thank you very much!
Nanoflow LC-MS (low-flow chromatography combined with High-resolution MS) would be a gold standard for this purpose. There are many applications for single-cell proteomics (SCP) that can detect peptides/proteins in very low amounts. You may extract the signature peptides through databases and may follow the abundances of these peptides with SRM mode acquisition (developed method by Skyline) or at the SIM level to estimate the abundance of the target protein...
Good luck,
Emir
Sebastian Schmitt
would it be possible to detect hPar17 with an specific antibody even if it’s very low expressed in mitochondria (by immunostaining my HCM cells)? I wondered if staining the cells would be sensitive enough to detect the low amount of protein. My main goal is to identify potential binding partners of hPar17 in mitochondria.Thanks a lot!
Cell staining is possible, especially if you want to co-localize your protein. However, insufficient sensitivity will likely be an issue. MS would indicate presence of your protein, but that's about it. If I were you I would seriously consider protein tagging and/or overexprexsion to start off. You could then perform coIP, proximity based assays etc...Good luck!
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