4 replies to this topic

[scistudent]

Hi,

I'm having some problems with my western for a phosphorylated protein, the first time I did it on some old samples to test the antibody using PBS (by mistake) it worked better than it is now when using the TBS.... Do you always have to use TBS for a phospho antibody?? or is it just sometimes better? I'm down to my last bit of protein and still struggeling to get a band I can do densitometry on.. any ideas?

thanks!

[AussieUSA]

Although some people say PBS will interfere in the specificity of the phospho-specific antibody, it is really dependent on your antibody. If the TBS is causing problems, consider adding tween 20 (0.05%) to the TBS, to help eliminate background problems.

Ultimately, do what works for you and your western.

AussieUSA.

[bob1]

Also, if using an alkaline phosphatase conjugated 2˚ antibody, use TBS, PBs will interfere with the phosphatase.

[miBunny]

Some antibodies really have a preference for staining in either PBS or TBS.  If an antibody is not staining well, this is one of the things I always test.

Edited by miBunny, 06 October 2009 - 05:51 PM.

[scistudent]

Great thank you! I will try with PBS now, I'm using LiCor antibodies so the PBS shouldn't interfer I think. THanks for the help!