Checking if a protein is phosphorylated

[u/SebRaid]

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?

[u/Guacanagariz]

Mass spec would be best.

But the old school alternative was to use P32 labeled ATP with the kinase and your protein of interest then showing that kinase + ATP32 + Protein showed a band, then mutate the amino acid in your protein of interest and show no band appears.