I am performing an in vitro kinase assay with a kinase and its substrate proteins. When I setup the assay and ran using 8% SDS-PAGE gels, I could see the samples only getting stacked but not separated. Moreover, the samples are stacked inside the wells itself. I tried using multiple buffers and different % of gels, but could not separate the samples.
The reaction condition what i have used contains 20 mM Tris-HCl, pH 7.5, 5 mM MnCl2, 5 mM MgCl2 and 1 mM DTT.
The proteins were incubated in the reaction buffer with 5 microCi of gamma 32P ATP for 30 min at room temperature and then stopped the reactions by adding 6X sample loading buffer and denatured at 95C for 10 min. The samples were further resolved in 8% SDS PAGE gel, dried and exposed to X-ray films.
If I understand you correctly, you run a protein gel but all the sample is stuck in the wells. You've tried a few types of gels but the sample is still stuck in the wells. It sounds like there is a problem with your protein preparation. First, can you separate protein from the non-kinase-assay sample (i.e. can you separate the "substrate proteins")? If not, you should consider that your protein prep is leaving you with "gummy" proteins that can't be separated. If you can separate whatever you're using as substrate (as assessed by Coomassie or other staining), then there's a problem with your assay mixture. One thing that looks a bit suspect is 10 minutes at 95oC for denaturation - you might be boiling down your sample to a thick goop.
You could also try to use phosphate-affinity SDS-PAGE using Phos-tag to detect mobility shift in proteins after phosphorylation or dephosphosrylation.
If I understand you correctly, you run a protein gel but all the sample is stuck in the wells. You've tried a few types of gels but the sample is still stuck in the wells. It sounds like there is a problem with your protein preparation. First, can you separate protein from the non-kinase-assay sample (i.e. can you separate the "substrate proteins")? If not, you should consider that your protein prep is leaving you with "gummy" proteins that can't be separated. If you can separate whatever you're using as substrate (as assessed by Coomassie or other staining), then there's a problem with your assay mixture. One thing that looks a bit suspect is 10 minutes at 95oC for denaturation - you might be boiling down your sample to a thick goop.
I agree with Lori's comments, either you are turning your protein into gummy bears with the heat or you are simply loading too much protein and it gets sticky. Try denaturing with 8M UREA without boiling.
Good luck,
I can very well separate the proteins without gamma p32 (CBB staining). I suspect the addition of p32 is doing some modifications, so that the proteins can't separate in the gel. Also i tried with 95C for 5 min. Still I couldn't separate the proteins.
To get you right: You can separate your proteins if doing the kinase assay with "normal" ATP + CBB-detection but not with radioactive ATP + X-ray-film? Did you try the kinase assay with 32P-ATP + CBB? Did you fix your radioactive gels before drying? Maybe the problem lies within the detection and not in the separation.
No I haven't try 32P-ATP+CBB. Yes! I dried the gels and loaded in the X-ray cassette.
The problem is in separation, because I could see the bands at the end of the stacking gel.
Typically pictures are shown in the same vertical direction as the run - are you sure, your look at the right orientation? Maybe the "dark line" in the lower part of the picture is the free 32P-ATP which is not detectable by CBB staining but of course gives strong signals in X-ray detection. You get nice bands of your labeled protein above, so there is definitely a separation and not all is stuck.
But I am getting bands at the end of the stacking gel. This is been verified with protein ladder
Hi there,
Some protein sample will render a smeary profile (or even stay in the well) on a SDS gel after 5min boiling at 95deg and will run "normally" when the protein sample is not boiled at all. This is rare, but it happens. This is maybe something to consider after you tested previous answers above.
I have experienced what Maxime mentioned. When you run the non-kinased protein substrates on a gel, do you boil those and in the same sample buffer as the kinased samples? Or, are you only boiling to stop the kinase reaction. I have had in vitro reactions or cell lysates that I have had to heat to only 42 C for 10 min to solubilize or else the protein I was looking for would not get into the well or past the stacking gel:resolving gel interface.
Thank you maxime and christopher. Let me try without boiling the samples
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