:: Search forums :: Top Users :: Reagent
Position: Discussion forums > Bioscience biotechniques discussion forums > Proteomics and protein biochemistry forum
|
can only detect overexpressed, but not endogenous, protein?
Posted by: wbd2975 (IP Hidden, New member, 5)
Date: May 10, 2005 04:02PM
Has anyone out there had the problem of only being able to detect overexpressed protein, but not endogenous levels in cells which should produce (albeit low amounts of) the protein, on Westerns? If so, what manipulations, if any, allowed detection of endogenous protein? I've tried standard troubleshooting (different Ab concentrations, different masses of loaded protein, different lysis buffers, different lysis protocols).
Any ideas would be much appreciated, Brian
Re: can only detect overexpressed, but not endogenous, protein?
Posted by: erik (IP Hidden, Unregistered user, )
Date: May 10, 2005 05:28PM
hi, maybe I didn't get you. Are you trying to detect both endogenous and overexpressed protein with exactly the same migration pattern in a denaturing gel? meaning that there is no any sequence differency between two proteins? If it is your intention, I don't think you can distinguish them by using antibody against the protein on a western.
Re: can only detect overexpressed, but not endogenous, protein?
Posted by: wbd2975 (IP Hidden, New member, 5)
Date: May 10, 2005 06:01PM
Sorry. My post wasn't clear enough.
The overexpressed protein and endogenous protein are in two different lysates (COS cells transfected with overexpression construct or human colon cancer cells that should have low expression) and, consequently, two different lanes of the gel. I can see the overexpressed protein, but not the endogenous. Brian
Re: can only detect overexpressed, but not endogenous, protein?
Posted by: erik (IP Hidden, Unregistered user, )
Date: May 10, 2005 08:42PM
hi, I guess you can try to use a very sensitive Chemiluminescent detection system such as ECL plus or SuperSignal West Pico Chemiluminescent from Pierce. Both are good at picking up signals from low abundant proteins. Good luck.
Re: can only detect overexpressed, but not endogenous, protein?
Posted by: femmeauburn (IP Hidden, Advanced member, 115)
Date: May 12, 2005 12:07PM
Keep in mind though that if the endogenous protein you wish to detect makes up less than 0.2% of the total protein in the sample, signal enhancement by ECL plus or SuperSignal may lead to the appearance of artifical bands. Do you know the endogenous relative concentration of the antigen of interest?
Re: can only detect overexpressed, but not endogenous, protein?
Posted by: wbd2975 (IP Hidden, New member, 5)
Date: May 12, 2005 04:43PM
Unfortunately I don't know the endogenous concentration of this protein, although I know it is low in these cells (using data done by other people in which they compared different cell lines).
I have already tried the pico and femtomolar ECL systems, and this has not helped. In fact, I already see other (presumably) non-specific bands, some of which are close to the molecular weight of this protein. Of course, one obvious explanation is that one of these bands is the protein I'm looking for. The problem is that this interpretation is completely inconsistent with the known biology of this protein, because the behavior of this band does not follow the pattern of other work (not done by me). Basically, I am trying to think of every possible technical problem that could explain this result before I begin concluding that the other (published) work is wrong. That's why I'm exploring the possibility that none of these bands is actually my protein. The problem is that my overexpressed positive control is actually the mouse ortholog of this human protein, which although quite similar, is slightly larger and therefore not a perfect control. I am working on getting a human construct. I am also working on looking at RNA levels to verify that the underlying biology is right. But I still need to visualize the protein. The only alternative conclusion I can come up with is that this protein is of such low abundance and the antibodies we have are insensitive enough that I am simply not detecting the protein in these cells. One approach I'm trying is to just use nuclear protein, as this protein is nuclear, to enrich for its presence. Also, the protein is known to be quickly degraded, so I wonder if the kind of lysis buffer or protease inhibitors might make a difference. But before I go throwing stones at other data, I want to try everything on my end. So any other suggestions are welcome. Thanks, Brian
Re: can only detect overexpressed, but not endogenous, protein?
Posted by: femmeauburn (IP Hidden, Advanced member, 115)
Date: May 13, 2005 10:27AM
sounds like a good approach to try enriching nuclear proteins only. i agree with you that the protein is probably of too low abundance to be detected. BTW: the molecular weight proteins that you mentioned were close to your protein of interest, were they of a lower molecular weight? If so you may be seeing proteolytic breakdown of your antigen. You can solve this by adding protease inhibitors such as PMSF, Pepstatin, or leupeptin. A lit search might tell you which inhibitor would be best for your samples.
Re: can only detect overexpressed, but not endogenous, protein?
Posted by: wbd2975 (IP Hidden, New member, 5)
Date: May 13, 2005 12:11PM
There are bands which are both higher and lower than the predicted molecular weight, and I do already use protease inhibitors. However, it is a good idea to figure out which inhibitor(s) would be best. I will search the literature.
Thanks for the suggestion.
Re: can only detect overexpressed, but not endogenous, protein?
Posted by: femmeauburn (IP Hidden, Advanced member, 115)
Date: May 13, 2005 02:17PM
No problem. I understand you wanting to check everything out before challenging the current studies. Hopefully enriching the nuclear proteins will give you the correct (specific) band. if you already use protease inhibitors the lower weight bands are probably artefactual "ghost bands" as well, unless the inhibitor you use is not correct for your samples. Good Luck!
|
We are moving ... Please post to our new community forums
