What are the most common mistakes done with secondary antibodies?

Secondary antibodies are the crucial reagents which allow visualization of primary antibody binding to a given target protein. Used in variety of techniques, including Western blot, immunolocalization, immunoprecipitation, to name a few. Incorrect secondary antibody can lead to increased background, poor reproducibility and non-specifi bands. Over the years, we have learnt that the most common mistakes done with secondary antibodies may include:

Too long incubation times, which may lead to increase background. Most optimal is incubation for 30 minutes to 1 h at room temperature.
Use of secondary antibodies not diluted enough, for example 1: 1000 instead of 1: 10 000, which may lead to increased background
Using secondary antibody which is not matching the host, anti-rabbit for antibody made in mouse, for example.
In case of monoclonal antibodies, it is crucial, that a secondary antibdoy is matching the specific class, primary monoclonal antibody is, for example mouse IgG1, IgG2b
Re-using of secondary antibody solutions
Incorrect storage which may lead to aggregates and decrease secondary antibody performance
Using secondary antibody which does not match detection method, for example ALP conjugated for chemiluminescent detection

We recommend to always read catefully product information sheet before antibody is purchased and afterwards, to apply correct dilution and storage conditions. These information should be always included on secondary antibody product information sheet.

How secondary antibodies work

Agrisera Highly Efficient and Most Popular Secondary Antibodies

What are the most common mistakes done with secondary antibodies?

Technical tips

Latest

Archive