After proteomics analysis of my samples, a specific phosphorylation site on a kinase was found. But when I want to validate the result I could not find any commercial ab to that phosphorylated amino acid. What can I do?
Hi Anita,
you could try an immunoprecipitation experiments where you pull-down your protein with a specific antibody via protein-G, than separate the precipitated fraction by SDS-Page and do a western blot with an anti-pTyr antibody.
It also works the other way around (pull-down with anti-pTyr and western blot with the specific antibody against your protein).
Hope this suggestion helps?
All the best with your experiments,
Christian
Is the specific phosphorylation observed at tyrosin or a different amino acid is phosphorylated?
Generally speaking, using MS-based omic is sufficient to validate PTM identification...
If protein level validation is needed, the suggestion given above is adequate if the phosphorylation is at tyrosine residue...
If peptide level site-specific PTM must be verified, other than MS-based analysis you may use dot blot or ELISA assays after peptide fractionation and conjugation with the purposed peptide to an anti-p antibody to conclude if a specific antibody is found...
For the protein level validation PTM PTM-specific staining may also be OK after PAGE application but site-specific information is lost indeed...
You may use phosphatase treatment to remove phosphate moieties and next you may observe the mass shifts to prove the PTM and its bound peptide location...
Last but not least; you need to use ETD alternative fragmentation to get c and z ions to validate the phosphorylation site (amino acid level) and this may exclude the need for any antibody-based approaches or any further validation...
Good luck, İEA...
Hi, Anita, To get aligned; if your kinase target is phosphorylated only through the serine residues and if you do not have any antibody against your kinase (not targeting the phosphate residues but searching for any other peptide sequence of your kinase as epitope to bind), using anti-p-tyr antibody would be not effective indeed....But other than serine, if you have other phosphorylation sites at your kinase, using anti-p antibodies can also indicate the knock down/gene silencing experiment is working properly... Anyway, without any specific antibody, you may not conduct ELISA or immunoblot assays, unfortunately...I mean if your kinase has merely serine-linked phosphates and there is not any anti-ser-p antibody or anti-kinase antibody you should focus on other techniques such as MS... In this case, if you cannot raise your specific antibody, it is better to follow the MS multiplexing experiment procedure. Relative quantification would be a nice option for certain validation...There are a couple of multiplexing reagents for relative quantification on the market. Isobaric labeling of the kinase peptides on their N terminus after enzymatic digestion and pooling the samples including treated and untreated will be the first step. Secondly comparing the obtained spectra of the phosphorylated serin existing peptide will tell you the fold change of the target quantitatively... Alternatively, You may link the transcriptomic outputs with the MS results also...RNA level suppression will enlighten the protein level decrease and possibly show a good and meaningful correlation... I am not sure but phospho-staining visualization after PAGE application can also give something about kinase downregulation since used dyes target phosphates non-specific to region or AA. Phos-tag SDS-PAGE is a phosphate-affinity electrophoresis technique that separates phosphorylated and non-phosphorylated proteins using conventional SDS-PAGE. Thus, a decrease in band intensity can reflect the downregulated phosphorylation at kinase and/or downregulated kinase expression...
If it's phosphotyrosine, immunoprecipitation followed by detection with an antibody like clone PY20 should work. For pSer and pThr, you'd need a context specific antibody. If you're lucky, the motif appears in other proteins (which kinase is phosporylating the protein?) and you might find a commercial antibody. Otherwise, You'd need to generate your own with a synthetic phosphopeptide.
Hi Anita,
there are several Anti-Phosphoserine antibodies available. The IP procedure is the same for all antibody-couples.
Best regards,
Christian
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