For epitope mapping, does the antibody need to be pure?
We recommend Protein A purification of antibodies for epitope mapping. However, the major component of crude serum or ascites fluid is serum albumin, which is used in the blocking step anyway. Following the direct coating method in the Manual, you can coat with serum or ascites fluid (diluted 1:10 in TBS) and omit the blocking step. If using the Protein A/Protein G bead capture protocol, use 1 L of serum or ascites fluid in place of the antibody in Step 5. In this case it is necessary to carry out the blocking step (Step 4) as described. However, given the variable level of antibody in serum or ascites, we cannot guarantee the crude antibody preps will work.
