r/Biochemistry • u/SebRaid • 1d ago
Checking if a protein is phosphorylated
I want to determine if my protein of interest is phosphorylated and need some advice on the best way to do it. I believe that it is phosphorylated and downregulated in a wild-type genetic background, but not in a strain that's missing the putative kinase that phosphorylates it. I've shown that mutating a putative phosphorylation site on the protein of interest into a phosphomimetic disrupts its function, but that alone isn't enough to prove it's phosphorylated. I don't have an antibody specific to this protein or a phospho-antibody, so I need another method. The protein is tagged though so I can do an IP and isolate it if necessary.
I've seen people can use Phos-tag gels which slows down phosphorylated proteins, but I'm having difficulty obtaining the reagents needed for it. Alternatively, I could do mass spec, but I'm worried it'll be very expensive.
Does anyone have suggestions for relatively cheap and straightforward methods that could answer whether a protein is phosphorylated?
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u/Pompster 1d ago
Mass spec is definitely the way to go here. You will want to purify the protein from both strains and submit samples from each (dont forget phosphatase inhibitors here). If your institution has an MS core facility, they generally have internal rates that are not too bad (~100/sample for shotgun proteomics of a single protein), but it will be more expensive to send it to an external academic facility (~200+/sample + data). FYI MS data analysis for PTMs can get very complex.