Hello,
I am trying to determine the multimeric state of my protein as literature suggests it exists as monomer, dimer, tertramer, etc. based on its function. I isolated these proteins from hek293T using RIPA buffer (it did not denayture my protein). I have detected the monomeric state of this protein through SDS PAGE and Western blot and now I am trying to do the blue native PAGE protocol using 4-6% Bis-tris gel from Thermo. I also used their sample prep kit to prepare my samples. I cut the gel into half, one was coomassie stained while the other was blotted onto a PVDF membrane. After blotting, only the coomassie dye from the blue cathode running buffer got transferred to the membrane, while after staining of the other half of the gel, I could see my protein stuck in the wells. I used NativeMark unstained marker and did not detect any bands. I'm not sure what is wrong. Please help.
You do not say, whether your gel Coomassie-stained showed any bands, besides the wells. Also, if you stain the gel after blotting from it, do you see bands?
If you do not see bands, then our proteins may have passed through the blotting membrane. Try using a stack of 2 or 3 membranes, instead of just one Another option would be to play with the blotting time, to reduce it.
If you can see any bands in the gel used for blotting, then you may try increasing the time for blotting.
Hi Achim Recktenwald, the coomassie stained gel did not have any bands as well, only the dark blue on the wells so I know the probem came from the gel run.
Are you sure that your sample buffer has the right amount of SDS in it?
What is the molecular weight of the protein? Are you perhaps using a gel with too small pores?
Achim Recktenwald I try to avoid SDS as I'm worried it will denature my proteins.The only SDS there came from RIPA buffer which was 0.1%. As a monomer my protein is 47kDA but it exists as multimers and how many monomers in one multimer is what im
Should have read your post more carefully.
If your protein is not moving in a native PAGE, then it is likely that the pI of your protein is too close to the pH of the buffer system; the protein will not have sufficient charge to move in the electric filed in the allotted time for your blotting procedure.
You could try what happens, if you double or triple the time for blotting or if you can increase the Voltage. But with the latter be careful that you do not overheat your protein or something goes up in flames.
Depending on the protein's charge, it may also run into the other direction. try a blotting procedure with a membrane, better a stack of 2 to 3 membranes on the other side of the gel.
Do you know your protein's pI? The theoretical pI may give you an indication, but a measured one by running an IEF gel would be better. Depending on the result, you may have to change the buffer system for the blotting process. Try to have at least a 2 pH-unit difference between pI and buffer pH.
Achim Recktenwald you might be right regardin the pI. The theoretical pI is 6 and the buffer pH is 6.8. Do you think I should try a more basic pH?
It is a long time ago that I did protein blotting, but there are tested buffer systems published for such protein's that do not fit in the normal scheme. If I remember correctly, the "standard" blotting assumes the protein's pI to be higher than the buffer pH. Assuming I am correct, than you would need a more acidic buffer.
What is the buffer compound used in your blotting buffer?
Achim Recktenwald oh I thought we were still talking about the gel run. I have identified the problem to be during the Native PAGE run itself. So I think I should mess with the running buffer or the sample prep. Sorry for the misunderstanding.
beulah mae ann villalobos Solivio , I am trying to get a blue Native gel to work as well, for a similar purpose. Any progress? I have no bands in any of my gels!
Wendy Pippin I tried adding digitonin and DDM to my samples and I was able to see some bands during the run. When I transferred the protein to the PVDF membrane all the coomassie went to the membrane as well and interfered with my detection so I can't be sure the bands were my protein of interest. I contacted thermo tech support but all they said was my sample was not compatible with that system. Now I am trying to lyse my cells using their 4x Native PAGE sample buffer and digitonin or DDM as suggested by their manual. I will give an update how the results turn out.
Wendy Pippin it's not even a membrane protein, which I understand are harder to characterize.
mine isn't either...i only asked because of the digitonin and DDM. those are for solubility? I have not seen those in some of the Native PAGE protocols I've been looking at. I have read that if protein stays at the top of the wells, there might be protein aggregation, so it may be worth using small amounts of DTT or 2ME, which shouldn't disrupt the subunit bonds, although I have not tried this yet. :-{
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