Dear colleagues,
I am doing Western Blot on mouse serum. I know that ELISA would be maybe a better choice but this was some sort of not my decision to choose Western Blot instead of ELISA:). I have a problem with albumins giving not specific, strong bands between 50 and 70 kDA which makes impossible to detect my protein of interest namely Angiopoietin 2 which should give a signal around 50-57 kDA. The signal from albumins is possibly due to unspecific binding of secondary antibody. As kits for albumins removal may not be efficient enough, Abcam recommended me using pre-absorbed goat anti-rabbit secondary antibody (ab7090) (primary antibody is PA5-27297). In case of pre-absorbed secondary antibody not specific reactions with proteins in mouse tissue should be strongly reduced. Have you got any experience with that kind of secondary antibody? I would not like to buy a pig in a poke:).
I would be grateful for your answers/ideas:),
Ewa
I prefer using biotinylated antibodies. I then detect with avidin-HRP. Has solved a lot of non-specific secondary antibody problems that I had. Know there are biotinylated anti-mouse angiopoietin commercially available. Check biocompare web sit for antibodies.
Also, are you using a diluted mouse serum or a "neat" serum sample to load on your gels? If you have a mouse serum sample that you can practice with - perhaps you can try a range of dilutions (adding PBS to your sample). That way, you might perhaps avoid "strong bands between 50 anf 70kDa" for albumin and get only one (hopefully, a sharp one) band instead - that may be very close to 70kDa. Then, if you run a 6% gel or a grading gel, you may be able to separate your desired band of 50-57kDa from the albumin band.
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