They are very large differences regarding concentration of cold ATP in these assays. Some people use high excess of cold ATP relative to the concentration of radiolabeled ATP, some people don't use cold ATP at all.
My second question concerns the unbound radiolabeled ATP on gel. How to get rid of it? As you can see on attached photo band corresponding to labeled histidine kinase (indicated with an arrow) is close to the unbound gamma ATP zone and therefore is less legible. I used Schager von Jagov electrophoresis method with 10% separating gel.
The ratio of cold to hot ATP is going to affect detection sensitivity; the wide variation in conditions may therefore be related to different concentrations the target protein i.e. a protein at very low concentration in the sample may not be detectable if you have too much competing cold ATP. If you can see the band of interest then you have the appropriate amount of cold:hot ATP. The sample itself could conceivably contain cold ATP (originating from the cells) but that will depend on how the sample has been prepared.
You can always run the gel for longer to allow the hot ATP to run off the end, but you risk losing small proteins too. If you are able to use a gradient gel it may prevent the bands of interest from migrating too far, while allowing the ATP to be cleared from the end of the gel.
Protein kinase activity is measured most conveniently using radiolabeled [γ-32P]ATP and an appropriate acceptor peptide or protein substrate. This method can be used to study the phosphorylation of all proteins that are phosphorylated on serine, threonine and tyrosine residues, except the extremely few proteins that would not be positively charged at a pH of 1.8. Small peptide substrates should have a charge of at least +2 (and ideally +3) at a pH of 1.8 to bind quantitatively to phosphocellulose paper in the assay conditions described here. The addition of basic residues to the N or C terminus of peptides that are not positively charged at a pH of 1.8 circumvents that problem. Typically two lysine residues are added to the N terminus of the peptide. The number of acidic residues in a peptide substrate sequence is of no consequence, as they become uncharged at a pH of 1.8. Phosphorylated histidine is labile in acid and protein histidine kinases must be assayed by different procedures.
http://www.nature.com/nprot/journal/v1/n2/full/nprot.2006.149.
Hi Mariusz Madej , I agree with Nick Gee answer .. good luck.
Thank you very much for your answers! Actually, at the beginning I didn’t use cold ATP at all (the photo shows the result without cold ATP). Then I tried to use cold ATP at concentration 500uM (the concentration that I found in publications) and the signal disappeared. My question is, why we actually use cold ATP? To prevent (or limit) unspecific binding of hot ATP? There is one unspecific band higher than specific one on my gel, which can be problematic in phosphotransfer, which I plan in the future. Do you think that I should check different concentration of cold ATP to optimize the specific to nonspecific signal ratio?
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