gel and monoclonal antibody issues - (Jul/08/2008 )
Hello,
My first problem for today is band width. After developing my blot, I can see that some of lanes contain bands that are wider than others. I run a 7.5% gel. While making the gel, I swirl the tube after the addition of each component. I do not vortex. I did notice that when I removed my comb from the stacking gel I was unable to totally clean out my wells. they still had some residual "wisps" of gel floating in them. To clean my wells I use a transfer pipet and squeeze running buffer into each well to force any gunk out. Could this be the problem? Do you know of any other issues that may cause one lane to run wider than another?
My second problem deals with my monoclonal FLAG:HRP antibody. In the past I have done an IP with a polyclonal FLAG and then blotted the membrane with a monoclonal FLAG:HRP. Those blots come out clean, with my empty vector lane showing no band. I recently IPd with a phosphotyrosine antibody and then probed with FLAG:HRP. This time my empty vector lane has a band exactly where it would if it had been transfected with my protein. There are also 3 other bands visible in each lane that should not be there. I thought that monoclonal antibodys were supposed to exclusively interact with only their target protein. I don't know why my monoclonal FLAG:HRP is interacting non-specifically with other proteins.
Please help! Thanks!
You are correct, the monoclonal antibody IS supposed to interact ONLY with it's target. With antibodies there is always some non-specific binding but this is so insignificant that you should NOT detact the antibody if the antigen is not present...
The odd thing is that the stain shows up exactly where it should be IF the target were there... I don't think the problem is with your anti-bodies...
This IS an odd problem... Have you run the protocol more than once?
To save time, and eliminate experimental error as far the gels are concerned, I purchase premade gels. They're not terribly expensive and it saves a great deal of time. How about trying a pre-made gel just to eliminate the possibility that something is wrong with your gel...
The odd thing is that the stain shows up exactly where it should be IF the target were there... I don't think the problem is with your anti-bodies...
This IS an odd problem... Have you run the protocol more than once?
To save time, and eliminate experimental error as far the gels are concerned, I purchase premade gels. They're not terribly expensive and it saves a great deal of time. How about trying a pre-made gel just to eliminate the possibility that something is wrong with your gel...
I have only run this particular protocol once. I have done very similar protocols but with different IP antibodies. As I mentioned before, when I IP with a polyclonal FLAG and blot with the monoclonal FLAG, the results are exactly as expected. I know that a phosphotyrosine IP would result in pulling down many different proteins. Is it possible that some endogenous proteins would interact with the monoclonal FLAG? I use my monoclonal antibody at a dilution if 1:1000 in TBST.
Also, does anyone know of any mistakes that could happen during an IP that cold result in a monoclonal antibody recogizing a non-specific protein?
If you only do a WB with your monoclonal AB, do you have a lot of bands? And have you tried IP with phospho-Y and blotting with your polyclonal? If so, are the results expected?
Maybe your monoclonal is not that specific. It would not be the first time an antibody has trouble with its specificity ^^