Nonspecific Binding on Western Blot
Posted 02 November 2004 - 11:35 AM
I am getting a lot of non-specific bands that show up on my western blot .
I am using an anti-his monoclonal antibody that is verified to give almost no background. I am using a goat anti-mouse polycolonal for my secondary Ab.
When I do the HRP detection, I get a lot of non-specific bands that show up on all samples (including the negative control and positive control) and I can't tell if my protein of interest is one of them or not.
I am blocking with 5% dry milk and incubating the antibodies in blocking buffer as well. I am also washing the membrane pretty good. Any suggestions? thanks
Posted 03 November 2004 - 10:21 AM
What is your sample? Is it lysate from eukaryotic cells? Anti-His Ab can has some non-specific binding with eukaryotic proteins. You can try titrating of loading of your sample. And as well play with your anti-His Ab dilution. Try to find lowest amount of your sample for which you still have a signal, very often non-specific band will disappear at this conditions.
Posted 04 November 2004 - 10:49 AM
Posted 14 August 2009 - 07:44 AM
So I guess the lesson is, no BME if you are going to be doing a western. Use DTT instead.
Posted 14 August 2009 - 05:53 PM
Have you tried different blocking agents (5% BSA or 3 % ova)?
Have you tried switching PBS for TBS (or vice versa)?
Do you have tween in your wash and block buffers?