Using Secondary Antibodies that have been Optimized for Western Blotting

This article introduces the HRP and AP conjugated secondary antibodies that have been optimized for western blotting.

Western blotting is known to be among the most sensitive of laboratory methods for the detection of a specific protein. The last step in many experiments is to visualize the specific protein, using a secondary antibody that has been labeled to bind it.

HRP secondary antibodies are beneficial in this role compared to primary conjugated antibodies in that either HRP or AP secondary antibodies confer significant increases in the sensitivity. This is due to the fact that both HRP and AP conjugated secondary antibodies can produce primary antibody binding at multiple sites, so that the same primary antibody can be bound to more than one HRP secondary antibody. Each of these secondary antibodies carries several HRP or AP labels.

These HRP or AP secondary antibodies are therefore well-suited to western blotting as they can amplify the signal produced, and can make it easier to detect the protein of interest when it is part of a complex mixture of proteins.

Biotinylated secondary antibodies are commonly used in immunohistochemistry.

The following is a list of HRP conjugated secondary antibodies that have been optimized for use in western blotting:

  • Goat Anti-Rabbit IgG (HRP) (ab205718)
  • Goat Anti-Mouse IgG (HRP) (ab205719)
  • Goat Anti-Rat IgG H&L (HRP) (ab205720)
  • Donkey Anti-Mouse IgG H&L (HRP) (ab205724)
  • Anti-Rabbit IgG VHH Single Domain Antibody (HRP) (ab191866)

Why Should You Use HRP and AP Secondary Antibodies that Have Been Optimized for Western Blotting?

In order to obtain assured results from western blotting, Abcam has a range of stringently tested HRP and AP secondary antibodies that are optimized for use in western blotting. They can be used after high dilution with the confidence that the results will have a low background and high signals.

Abcam HRP and AP secondary antibodies have been numerously mentioned in scientific literature.

AP conjugated antibodies may be used on a routine basis from any dilution within 1/5,000 to 1/50,000. With a 1/1,500 dilution, it is possible to carry out 1,500 blots using just one vial of AP conjugated secondary antibody.

With HRP secondary antibodies, the dilutions applied may be between 1/2,000 and 1/20,000. If the 1/2,000 dilution is performed in 3 ml of milk/BSA, one vial of HRP conjugated secondary antibody can be used for 600 blots.

About Abcam

Abcam is a global life sciences company providing highly validated antibodies and other binders and assays to the research and clinical communities to help advance the understanding of biology and causes of disease.

Abcam’s mission is to serve life scientists to help them achieve their mission faster by listening to their needs, continuously innovating and improving and by giving them the tools, data and experience they want. Abcam’s ambition is to become the most influential life science company for researchers worldwide.


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Last updated: Jun 6, 2019 at 12:03 PM

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