We have a mouse hybridoma cell line which can secrete a mouse IgG2a monoclonal antibody. The culture medium is RPMI 1640 medium + 10% horse serum + NEAA +
1 mM sodium pyruvate + penicillin (100 units/mL), streptomycin (10 ug/mL).
The purified antibody will be used in western blotting.
Question 1: Is that possible to use cell supernatant as "antibody" to blot? Can the 2nd antibody (anti-mouse) bind to IgG from horse serum or other mouse molecules because it is a mouse cell line?
Question 2: What is the best method or kit if I have to purify the antibody from tissue culture medium?
Thank you so much.
You need to know the class of antibody prior to purification (you can purchase an isotyping kit to do an Elisa). IgG can be purified using a commercial affinity chromatography column containing protein A or protein G. IgM can be purified by ammonium sulfate precipitation followed by removal of IgG with a protein A column.
However, Western blots can often be done using culture supernatants. This method depends upon whether your antibody is monoclonal and its specificity.
Thank you Diana! The antibody is IgG2a monoclonal antibody. Do you think the horse serum and other mouse molecules will produce a high background if I use culture supernatant to blot?
Usually it is OK, as your secondary antibody should bind only to mouse Ig, not to other molecules that may bind to the blot. However, that is one of those things that you may have to test using more than one blocking method to improve specificity.
If you choose to purify the antibody, I have attached a file that shows the binding properties of recombinant protein A.
Thank you so much! I will try the supernatant first and then go to purify if required.
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