Here are some factors to consider when selecting a secondary antibody for western blotting:

  • Host and target species: The secondary antibody should be raised in a species different from the host species of the primary antibody. For example, if your primary antibody is raised in a mouse, you will need an anti-mouse secondary antibody raised in a goat, rabbit, etc.
  • Targeted reactivity: The secondary antibody should be specific for the target protein that you are trying to detect. You can check the specificity of the secondary antibody by looking at the product information provided by the manufacturer.
  • Purification: The secondary antibody should be highly purified to avoid cross-reactivity with other proteins in the sample.
  • Cross-adsorption: The secondary antibody should be cross-adsorbed to remove any antibodies that cross-react with the primary antibody. This will help to improve the specificity of the assay.
  • Multiplexing: If you are planning to do multiplex western blotting, you will need to select secondary antibodies that are conjugated to different labels. This will allow you to detect multiple proteins in the same sample.

Once you have considered these factors, you can start to narrow down your choices. There are many different secondary antibodies available from different manufacturers, so it is important to compare the product information carefully to find the best one for your needs.

Here are some additional tips for selecting a secondary antibody:

  • Read the product information carefully: The product information should provide information on the host species, target species, targeted reactivity, purification, cross-adsorption, and multiplexing capabilities of the secondary antibody.
  • Ask for help: If you are not sure which secondary antibody to choose, you can ask for help from a technical support representative from the manufacturer.
  • Do a trial run: Once you have selected a secondary antibody, it is a good idea to do a trial run to test its performance. This will help to ensure that you get the results you want.