Optimized secondary antibodies for western blotting

HRP and AP conjugated secondary antibodies optimized  for western blots.

Western blotting is regarded as one of the most sensitive techniques to detect specific proteins; the final step of a experiment experiment is the visualization of specific proteins, with a labeled secondary antibody.

Using HRP secondary antibodies offers a significant advantage when compared to primary conjugated antibodies, since the use of HRP or AP secondary antibodies can greatly increase sensitivity. The increased sensitivity with HRP or AP secondary antibodies is because these antibodies are designed to bind the primary antibody in more than one place, resulting in several HRP secondary antibodies being bound to the primary antibody, each with multiple HRP or AP labels.

The use of HRP secondary antibodies and AP secondary antibodies is therefore ideal for western blot since there use allows amplification of the signal and easier detection of protein of interest in the middle of a complex protein mixture.

In immunohistochemistry (IHC) biotinylated secondary antibodies are commonly used, you can learn more about the advantages of these secondary antibodies here.

HRP conjugated secondary antibodies optimized for western blot

Reasons to use HRP and AP secondary antibodies optimized for western blot

  • For guaranteed results in your western blot, our HRP and AP secondary antibodies are extensively tested and optimized for western blot and can be used at high dilutions with confidence of high signal and low background.
  • Cited within the scientific literature.
  • AP conjugated secondary antibodies can be routinely used at dilutions ranging from 1/5,000 to 1/50,000.  Based upon a 1/5,000 dilution you can perform 1,500 blots with a single vial of an AP conjugated secondary.
  • HRP secondary antibodies can be used at dilutions ranging from 1/2,000 to 1/20,000.  Assuming a 1/2,000 dilution in 3 mL milk/BSA, it is possible to perform 600 blots with a single vial of HRP-conjugated secondary.

  • Fluorescent western blot is increasingly used since it has several advantages. Our fluorescent western blot secondary antibodies, IRDye® secondaries  range offers a tested protocol for your experiments.
  • Read next when to use  fluorescent western blot.