Is my antibody specific? - (Jul/01/2008 )
Hello everybody!
I am trying to see if the new antibody I received is specific for "my" protein. I have run a western blot with a E.coli-lysate and 2 different antibody quantities (1 and 0.2 ug). I alway run a control with just E. coli.
In the lane with the sample, there is a big double band at the right size, that should be my protein (even if the band shouldn't be double...). I tested it with an anti-his antibody directed against the his-tag of the expressed protein.
What are the other bands? Would you say that the antibody is specific enough?
Thank you very much!
[attachment=4918:antibody_specifity.jpg]
-LabS-
QUOTE (LabS @ Jul 1 2008, 05:24 AM)
Hello everybody!
I am trying to see if the new antibody I received is specific for "my" protein. I have run a western blot with a E.coli-lysate and 2 different antibody quantities (1 and 0.2 ug). I alway run a control with just E. coli.
In the lane with the sample, there is a big double band at the right size, that should be my protein (even if the band shouldn't be double...). I tested it with an anti-his antibody directed against the his-tag of the expressed protein.
What are the other bands? Would you say that the antibody is specific enough?
Thank you very much!
[attachment=4918:antibody_specifity.jpg]
I am trying to see if the new antibody I received is specific for "my" protein. I have run a western blot with a E.coli-lysate and 2 different antibody quantities (1 and 0.2 ug). I alway run a control with just E. coli.
In the lane with the sample, there is a big double band at the right size, that should be my protein (even if the band shouldn't be double...). I tested it with an anti-his antibody directed against the his-tag of the expressed protein.
What are the other bands? Would you say that the antibody is specific enough?
Thank you very much!
[attachment=4918:antibody_specifity.jpg]
Does the anti-his antibody also give you a doublet? Keep in mind, the doublet could be proteolytic processing of your protein which can result in multiple sizes (you can try using protease inhibitor cocktails to see if you can deminish this doublet). Depending on whether your His tag is N- or C- terminal, that could explain the doublet.
Also, its important to see whether you can increase the side of "your protein" by doing different induction times. If it is your expressed protein, it should go up significantly with time. Ex. 0 hr, 1 hr, 2 hr, 4 hr (post-induction) time points.
As for your antibody being specific enough: once you are certain that the protein band corresponds to your protein, you can always bring down the background bands by increasing blocking of the membrane and maybe using lower concentration of antibody or shorter incubation times. Keep in mind you can fine tune with different temperature conditions too. But, depending on the source of the antibody (especially when using polyclonal vs monoclonal antibodies), it is rather normal to see some LOW level cross reactivity.
Hope this helps,
Cheers!
-BioDoc-