Hi Shanshan,
With regards to you seeing multiple bands on your westerns, it is quite normal. That is occurring because most antibodies for western, with the exception of a few histone ones, are not monoclonal, so there were be background bands. Even quite a few monoclonal antibodies will recognize more than one epitope.
Also when working with tissues, you will have higher background due to the presence of antibodies against the animal used to generate the antibodies.
When we run westerns for epitope integrity, we cut the membrane such that it incorporates the area representing the size of the protein of interest. By doing that you reduce the amount of antibody you are using, but also render the binding to the region of interest more efficient.
Can you post your post IP bioanalyzer results as well as your western if possible?
Did you run the post IP DNA on a high sensitivity chip? If so, then the high sensitivity chip is very sensitive to salts, and will always give a higher size range and distribution as compared to the regular sensitivity chip. We are almost done with a technical note that clearly shows that and should have that available for posting by next week.
Thank you
hamid
With regards to you seeing multiple bands on your westerns, it is quite normal. That is occurring because most antibodies for western, with the exception of a few histone ones, are not monoclonal, so there were be background bands. Even quite a few monoclonal antibodies will recognize more than one epitope.
Also when working with tissues, you will have higher background due to the presence of antibodies against the animal used to generate the antibodies.
When we run westerns for epitope integrity, we cut the membrane such that it incorporates the area representing the size of the protein of interest. By doing that you reduce the amount of antibody you are using, but also render the binding to the region of interest more efficient.
Can you post your post IP bioanalyzer results as well as your western if possible?
Did you run the post IP DNA on a high sensitivity chip? If so, then the high sensitivity chip is very sensitive to salts, and will always give a higher size range and distribution as compared to the regular sensitivity chip. We are almost done with a technical note that clearly shows that and should have that available for posting by next week.
Thank you
hamid
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