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 IgG light and heavy chains of the primary antibody used for IP run at approximately 25 and 50 kD, respectively, on the subsequent western blot and can often obscure bands of proteins that have similar molecular weights. The use of antibodies from the same animal source for both the IP and the subsequent western blot is the cause of this result since the secondary antibody used in the western blot not only detects the native IgG used for the primary antibody incubation, but also detects the denatured heavy and light chains of the antibody used for the IP.  There are several methods to avoid this outcome (ranked from most to least reliable):

1. Use antibodies from different species for the IP and the western blot.  For example, use a rabbit antibody for the IP and a mouse antibody for the western blot, or vice versa. Note: The secondary antibodies need to be species-specific. Our testing has been performed with Anti-rabbit IgG, HRP-linked Antibody #7074 and Anti-mouse IgG, HRP-linked Antibody #7076, which are highly specific for rabbit and mouse, respectively. We have not observed species cross-reactivity with these secondaries (i.e., mouse reactivity with #7074 and rabbit reactivity with #7076). 
2. Use a biotinylated primary antibody from the same species for the western blot. The biotinylated antibody can then be detected with Streptavidin-HRP #3999.
3. If your target does not migrate near 25 kDa, a light chain specific secondary antibody can be used. We offer a Mouse Anti-Rabbit IgG (Light-Chain Specific) (D4W3E) mAb (HRP Conjugate) #93702 and a Rabbit Anti-Mouse IgG (Light Chain Specific) (D3V2A) mAb (HRP Conjugate) #58802.
4. Replace the secondary antibody in the western blot with our Protein A (HRP Conjugate) #12291. However, it is important to note that #12291 may cross-react with denatured IgG at high concentrations.
5. Use a conformation specific secondary antibody for the western blot. We offer a Mouse Anti-Rabbit IgG (Conformation Specific) (L27A9) mAb #5127.  However, it is important to note that #5127 may cross react with denatured IgG at high concentrations (similar to #12291).

Last updated: September 12, 2024