CST provides several products to avoid detection of IgG heavy chains. Which one is best for my IP experiment?

When performing immunoprecipitation (IP) followed by western blotting, the denatured immunoglobulin G (IgG) light and heavy chains of the primary antibody used for IP run at approximately 25 and 50 kilodaltons (kDa), respectively.

These denatured chains can often obscure bands of target proteins that have similar molecular weights on the subsequent western blot. This occurs because the secondary antibody used in the western blot not only detects the native IgG used for the primary antibody incubation, but also cross-reacts with the denatured heavy and light chains from the IP antibody when both are derived from the same animal source.

Methods to Avoid IgG Chain Detection

There are several strategies to prevent the detection of denatured IgG chains, ranked below from most to least reliable.

1. Different Species Antibodies: Use antibodies from different host species for the IP and the western blot (e.g., a rabbit antibody for the IP and a mouse antibody for the western blot, or vice versa). Ensure you use highly species-specific secondary antibodies, such as Anti-rabbit IgG, horseradish peroxidase (HRP)-linked Antibody #7074 and Anti-mouse IgG, HRP-linked Antibody #7076, to avoid cross-reactivity.
2. Biotinylated Primary Antibody: Use a biotinylated primary antibody from the same species for the western blot. This antibody can then be detected using Streptavidin-HRP #3999.
3. Light-Chain Specific Secondary Antibody: If your target protein does not migrate near 25 kDa, a light chain-specific secondary antibody can be used. We offer Mouse Anti-Rabbit IgG (Light-Chain Specific) (D4W3E) mAb (HRP Conjugate) #93702 and Rabbit Anti-Mouse IgG (Light Chain Specific) (D3V2A) mAb (HRP Conjugate) #58802.
4. Protein A (HRP Conjugate): Replace the standard secondary antibody in the western blot with Protein A (HRP Conjugate) #12291.
   1. Important: This product may still cross-react with denatured IgG at high concentrations.
5. Conformation Specific Secondary Antibody: Use a conformation-specific secondary antibody for the western blot, such as Mouse Anti-Rabbit IgG (Conformation Specific) (L27A9) mAb #5127.
   1. Important: Similar to Protein A, this antibody may also cross-react with denatured IgG at high concentrations.

Last updated: June 29, 2026