Hi,

Are you sure your pruification is exactly as the first time...?, maybe first time you were co purifying an intereactor necessary for protein activity, or your protein is somehow denetured. I would include a positive control using a general kinase substrate like MBP or Histones.

Hi,Antonio,the kinase autophosphorylation can be detect in the same degree from time to time.So,I think the kinase may not be the problem.However,it may be possible that kinase is the optimal substrate to itself ,so the autophosphorylation will not be affected.

Arrhenius,

Could it be that the phosphatase inhibitor or ATP is no longer active? I always make these fresh or from frozen stock. But check out this article (not by me).

http://www.ncbi.nlm.nih.gov/books/NBK91991/

Thomas

Repeated freeze/thawing may caused loss of enzyme activity. I would suggest a retrial with a fresh isolation as well as a positive control to determine where the problem lies.

Arrhenius,

First you should define what you call a "perfect result" and how you monitor substrate phosphorylation (32P, WB, ...). Then it's easier for everybody to figure out what went wrong.

Regards

My first trial include a Wild Type and a kinase dead mutant,the WB showes that Wild type can phosphorylate itself as well as the putative substrate,while the kinase dead mutant can't phosphorylate both.

I am sorry for not including these details in this question.

Thank you all!

Dear Arrhenius,

From your previous message I deduce that you work with recombinant protein kinases. Can you check the activity of your WT kinase on a synthetic peptide or another protein substrate ? Do you ave a positive control in your WB to check the phospho-specific primary antibody.You could then figure out whether the problem comes from the kinase activity or the WB.

Using a positive control, ie running a known kinas with a known substrate will help you determine if your reaagents are bad. Typically, ATP goes bad as the pyro phosphate bond hydrolysis. The stock solutions should be stored frozen, ie at least -20. I don't have experience ith pervanadate, but I thought that also needs to be prepared fresh. Maybe someone else can comment on the phosphatase inhibitors, and your enzyme can go bad during storage. So to trouble shoot, maybe you can prepare everything fresh? Also is your assay a Western blot? The comment by Didler is important, ie a positive control for the Western.

I just saw your results. It looked like the dead mutant had activity. Does the dead mutant have other phosphorylation sites. If it does, due to your prep possibly having a high enzyme activity, the dead mutant is being phophorylated. Enzymes, at high enough concentrations, with unnatural substrates, can do lots of weird things. Proteinases for example, will cleave unnatural substrates in vitro all the time if one uses enough enzyme or overexpresses them in cells. It does not mean it is physiologically relevant. But the enzymes will be kinetically competent to perform many types of reactions, and in your case, it would be phosphorylation at a different site. To overcome this, just use less enzyme, and better, change the substrate, so ther is only one phosphorylation site.

Very important point from Marcia. Use only catalytic amount of enzyme and try to have an idea of the phosphorylation stoichiometry and of any concomitant change of activity/function of your potential substrate. WB can be quite sensitive (but not quantitative) and a strong signal may only reflect a very low phosphorylation stoichiometry (< 1%). So pay attention to the (pato-)physiological relevance of the results.

@Didier

The assay is carried out with purified recombinant protein and both the autophosphorylation and phosphorylation of the substrate is probed with p-Tyr-100 as primary antibody.(Cell Signalling Technology). However I don't have a positive control at hand.From the articles I have read,I know the kinase can autophosphorylate,so I use it as a positive control.

@Marcia

The Dead mutant is unable to bind ATP in the kinase domain,so,should the dead mutant "autophosphorylation"be a background?I have tried once more these days with both the kinase and substrate freshly prepare just before the assay,so do most components in the kinase buffer except for DTT and ATP. DTT and ATP was diluted from 1M and 50mM stock respectively.However the result is still frustrating.

Thank all of you so much for your expertises,and i will try less enzyme next time and try to include a defined substrate in the assay.

I would use the kinase dead mutant auto phosphorylation and phosphorylation of substrate as background, if you are certain the mutant is completely dead. Sometimes mutants retain a low percent amount of activity. Thus, if you are using a large quantity of protein, and due to the sensitivity of the assay as Didier has noted, you may see some residual activity. As for phosphorylation of the Y to Phe mutant, the mutant substrate may be getting phophorylated somewhere else. So, I would try to standarize by protein assay how much protein enzyme and substrate you use each time. And I would maybe run an experiment with varying amounts of enzyme for both the dead and active proteins, in order to optimize your assay. The enzyme phosphorylation should be linear with time and enzyme concentration. Can y ou quantify phosphorylation with densitometry? I would do this.

@Arrhenius

One last thing you could try is to change your kinase buffer to this: 50mM Hepes or 10mM MOPS ph 7.3, 80mM KCl, 1mM EDTA, 1mM DTT, 5mM MgCl2, 1% glycérol and start with MgATP 300 uM. This is our all rounder kinase buffer that we have been using for years with S/T protein kinase.

Good luck

@Marcia

Indeed,I have tried a "dosage vs effect" assay,herein attached is the result.But I have not tried a time course study yet.The "dosage vs effect"assay was carried out with purified enzyme and substrate that was conserved in 4℃ for 48 hours,so I think I should tried it again with freshly prepare samples.

@Didier

Thank you for sharing me with your recipe.There is a quenstion in EDTA,should it chelate Mg in the buffer?Why is it included in the buffer constituent?Is it here for depletion of Ca ion?

Your results look great. And yes, I would think no EDTA as the magnesium is needed for the ATP to bind. But I am not an expert in kinase assays so I refer to you and Didier

@Arrhenius

EDTA will indeed chelate some Mg, but the latter is in large excess moreover my ATP stock solution is already prepared with Mg. EDTA is just there as a precaution to chelate any heavy metals that could "poison" your enzymes. For the buffer based on MOPS you can replace EDTA by EGTA 0.5mM but then be aware that some Ca2+ dependant kinases will be in trouble.