HELP!!! Non-specific binding on my membrane - (Jun/05/2011 )

Hi,

I'm new to western blot and has only started doing them on cell lysates for a couple of weeks. I've been using antibodies from Santa Cruz (IL-1b) and Enzo life Science (Nalp1 and Nalp3). The problem that I have with my membranes is that there are always non-specific bands even when I'm using monoclonal antibody. The bands that I'm getting never seems to be the ones that I'm after which are IL-1b (17kDa, 31kDa), Nalp1 (160kDa) and Nalp3 (117kDa).

I am using the invitrogen system. Products that I'm using are the NuPage Novex 4-12% Bis-Tris 1.5mm gels, NuPage MOPS SDS running buffer (20x) and NuPage transfer buffer (20x).

My membranes are being blocked in 5% Skim milk in TBST for an hour and primary antibodies incubated at the recommended dilution overnight at 4 degree celsius. I'm using Mouse and Rabbit HRP from R&D systems as my secondary antibody and using the ECL plus western blotting reagents from GE healthcare for imaging.

I know that the non-specific is not due to my secondary antibody because I've used it to detect b-actin and it looks awesome.

I need help and recommendations in how I can reduce the non-specific bindings and also if the primary antibodies that I'm currently using now is worth wasting time on.

Does anyone has any suggestions that I could use consider? I'm running out of options and need to present my results soon.

Try titrating your antibodies - start at the recommended dilution and work up from there. e.g. if you have 1:1000 as recommended, do 1:1500, 1:2000, 1:3000 as well, or something similar.

The bands that I'm after are really faint. Would further diluting my antibodies affect my results? Like would those faint bands of mine disappear?

So your specific bands are faint but the non-specific are strong? I was assuming the opposite situation.

If so, diluting the antibodies further won't work well. Have you tried blocking and diluting the antibodies in different compounds such as BSA?

bob1 on Thu Jun 9 09:39:05 2011 said:

Hi bob thanks for your suggestion. I'll try out your suggestion. We've just recently got the ECL advance western blotting kit with a blocking reagent within the kit itself. So I might try that as well and see how it goes. Not sure if the blocking would only be effective for background. Other than the non-specific bindings, my blot itself is actually quite clean.

In my experience, Santa Cruz antibodies are hit or miss. They have some great ones, but others are pretty bad. Is it possible for you to immunoprecipitate with a different antibody first, and then blot with the original one. That should help a great deal.

Otherwise, blocking in 5% BSA in TBST is the standard in our lab and works great. It sounds like maybe your target is just expressed at lower levels in the cell-type under investigation, so the non-specific bands become more dominant.

Hi:

If you're getting non-specific binding of your primary antibodies, shorten the time of incubation to 1 hour, instead of overnight.
Or, I've used a device from Millipore called the Snap i.d. system that uses vacuum to suck the antibodies and wash through the membrane after a very short (10-15 minute) incubation time. The blots were just as good as doing immunodetection the long way and the background was reduced.They have demo units to test. (http://www.millipore.com/life_sciences/flx4/western_blotting&tab1=5#tab1=5:tab2=2)