Antibodies Support—Troubleshooting

The definition of “didn’t work” depends on what you did with the antibody. For example, it could mean that you observed no specific immunofluorescence staining at the antibody concentration that we suggested. It could also mean that, at the antibody dilution we recommended, your western blots showed high background staining. The suggested antibody concentrations specified in our manuals should be considered starting points for further experimentation. These values were derived in our labs during development of the antibody, or they are based on the experiences of our collaborators as well as other customers. If our suggestions don’t work for you, try your method again with a different concentration/dilution. Continue optimizing until your results are as good as you can make them. If your experiments still don’t “work”, please contact Technical Support at techsupport@thermofisher.com for further help.

In all likelihood, your antibody stopped working because it lost its reactivity. This happens because antibodies (and most other proteins) are less stable at low concentrations (e.g., the μg/mL range and lower) than they are at higher concentrations. For example, proteins adsorb to surfaces like the walls of their containers due to charge-mediated and hydrophobic interactions. This occurs regardless of the protein concentration, and it usually results in some degree of protein denaturation and loss of activity. However, at low protein concentrations the impact of adsorption is larger per unit of time than at higher concentrations. Antibodies in solution also aggregate with each other for the same reasons that they adsorb to surfaces, sometimes resulting in loss of activity. How fast your diluted antibody loses activity in storage is unpredictable, so store your diluted antibodies no longer than overnight at 2–8°C and then discard them. Better yet, make a fresh working dilution each time you need to use the antibody.

Antibodies prepared against a short peptide sequence may not always recognize the full-length protein in which that peptide sequence is located. The peptide sequence only represents a small portion of the entire protein, and the full-length protein is usually a more complex structure with folds, α-helices, β-sheets, and other structural motifs as well as various posttranslational modifications, any of which can shield the epitope from the antibody. This is one reason it is important to read the manuals for our antibodies. They describe the capabilities and specificity of our antibodies and list the applications for which these products are validated.