Heavy/light chain contamination on Western Blot?

IPKine™ LCS antibody for target protein approaching 50 kDa and IPKine™ HCS antibody for target protein approaching 25 kDa.

25 kD or 50 kD protein detection on Western blots after Immunoprecipitation is often suffered from heavy or light chain blotting contamination. Abbkine’s IPKine™ secondary antibodies could solve these problems and bring you satisfying results with good performance.

Immunoprecipitation (IP) is the technique of precipitating a protein antigen out of solution using an antibody that specifically binds to that particular protein. The antigenantibody complex are precipitatied by affinity beads incubation. After the washing and centrifugating process, a western blot will be conducted to detect the target protein.

When anti-IgG (H+L) antibodies are applied to detect protein bands on Western blots after immunoprecipitation, two unexpected bands appear corresponding to the heavy (50 kDa) and light chains (25 kDa) of the precipitated primary antibody. These bands usually obscure detection of any protein of interest with a molecular weight near 50 kDa or 25 kDa. Abbkine’s IPKine™ products could solve these problems by eliminating heavy and light chains interference, seperately.

Below is good performance using IPKine™ HRP Mouse Anti-Rabbit IgG LCS to detect the NUP50 protein. 

Abbkine IPKine™ heavy chain secondary antibody reacts with Fc portion of mouse or rabbit IgG heavy chain, and it doesn’t react with the Fab portion of IgG. Its cross-reactivity with bovine, horse and human serum proteins has been specially minimized (anti-rabbit antibody only absorbed with human serum protein).

IPKine™ light chain reacts with kappa light chain on mouse/rabbit IgG. It doesn’t react with the heavy chain of IgG. Its cross-reactivity with human, rat, goat, sheep, bovine serum proteins has been specially minimized.

1. IPKine™ LCS antibody for target protein approaching 50 kDa

If the size of your target protein  is close to 50 kDa, in order to avoid interference of the antibody heavy chain , we recommend using light chain specific secondary antibodies.

2. IPKine™ HCS antibody for target protein approaching 25 kDa

In case your target protein is close to 25 kDa, we recommend using heavy chain specific secondary antibodies, which specifically bind only heavy chain of IgG thus to avoid IgG light chain interference.

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