Antisera vs. Antibodies
Posted 05 November 2009 - 07:18 AM
For some reason while I can get antibodies (and even cell culture sups from hybridomas) to stain nicely, I can never get anything but background/non specific staining when using anti-sera. This happened most recently using a commercially available antisera that other people have published staining with. I have this trouble with OCT embedded sections as well as tissue culture cells on coverslips.
Does anybody do anything different (or know of reasons to do anything different) for antisera vs. affinity purified antibodies?
Posted 06 November 2009 - 04:17 AM
Plus all normal proteins such as albumin, transferrin, complement, etc etc.
Affinity purified antisera will contain only the antibodies that react with the antigen; the antisera goes thru a purification processs where the antigen is on a column and the specific ab bind and then the column is washed and the antibodies bound to the antigen are eluted creating a specific affinity purified product (still polyclonal). There will be cross reacting antibodies in this mixture since it is polyclonal.
Your hybridoma will be monoclonal all the same antibody...depending upon how the original selection was made it can be very specific to the antigen or also have some cross reactivity as well.
Posted 09 November 2009 - 07:57 AM
Posted 09 November 2009 - 10:58 AM
You are (?) using a secondary ab-conjugate? Is it specific for the species IgG you have in your antisera; that is are the nonspecifics adsorbed out? If this conjugate cross reacts with non-IgG proteins of your antisera then you will have binding (it is specific since the ab recognizes proteins in addition to the IgG you are using for detection).
Thus, you have 2 things to consider: all the stuff in your antisera and the specificity of the 2nd ab-conjugate.