Determining whether the quality of an antibody and if it is species specific - (Sep/12/2010 )
I received a custom ordered antibody and need to confirm whether or not it is species specific. I am really new to this, but after some reading Iíve concluded that I should do either a Western blot or ELISA. Does anyone have a recommendation on which would be more definitive? If I run a Western, all I know is that I will need a negative control with serum and a blood sample containing the target protein, but would that be enough to show specificity? Should the blood sample be cleaned and the protein extracted first? Would I need to run samples from different animals, and if so, how many different animals? Or is this a standard that I can compare it to?
Thanks in advance from a very confused freshman!
What species specificity does the custom antibody have? What assays are you planning to use the antibody for? Do you simply want to make sure the antibody binds to the target protein?
By "species" do you mean "organism specific" or "protein specific"? If it is organism, then you need to get samples from several different species as many as you want, but if the antibody is against human protein you will probably want mouse, rat, rabbit, and goat. Make sure that you don't use the species in which the antibody was raised as you will just see IgG bands on that lane.
If it is protein, you should determine if the antibody is binding to the protein specifically by performing competition experiments where you incubate the antibody with a peptide corresponding to the epitope of the antibody and then see if it will bind to the target protein. You should titrate the amount of peptide used to show increasing competition. This is best done on western blots.