Frequently Asked Questions (FAQ)


Dr. Giorgio Cattoretti
Associate Professor of Clinical Pathology
Institute for Cancer Genetics, Columbia University
New York, USA




For how long should I incubate a primary antibody?



First a short introduction to the problem. Each antibody is generated through a T-cell dependent B-cell activation, affinity maturation in germinal centers, isotype switch and selection. Selection is by choosing animal, immunization schedule, affinity purification, performance in a particular assay (Western Blot, ELISA, IF, flow cytometry, paraffin section staining), choice of clone if monoclonal, lot if polyclonal. You may have to use an antibody which has not been selected for the use you want.


With a monoclonal antibody, affinity constant for the antigen and crossreactivity with unrelated antigens are fixed values, being the immunoglobulin clonal. If you have a monoclonal with high affinity, as little as 5 minutes may be enough. If you know you are dealing with high-affinity antibodies, stick to a practical half an hour (± time for coffee). However, we noticed negative staining with strong antibodies incubated for as much as one hour, compared with overnight incubation (e.g. p27, BCL6). This may be due to a combination of factors (Ag density, masking, affinity).

We use almost invariably high dilutions and overnight incubations. If the antibody is crossreactive with an unrelated clone at low affinity, long incubation time may allow unwanted binding, but at this point chose another clone or a polyclonal antibody. Concentration does not play a significant role.


Polyclonal antibodies represent a gaussian curve of various species (clones) with diverse affinity and crossreactivities. The relative proportion of the most antigen-avid and crossreactive-free clones makes a good or a bad antibody.

You can play with time and dilutions: very short incubations and relatively high concentrations will favor high affinity clones, even if not the most representative species. Long incubations and high dilutions will favor the most prevalent species, even if with low affinity.


Other considerations besides affinity may play a role in your choice of primary antibody staining duration:

  • antigen density. You may want to incubate overnight for <5K molecules/cell.

  • masking. When you do double staining, the first stain implies some degree of non-specific masking.

  • convenience. For research purposes, you can split over two days your tedious work.

  • Secondary antibodies are usually selected for high specificity and high affinity, so 45 minutes are usually OK.