For researchers who perform Western blotting following immunoprecipitation, antibodies specific for light chains or Fc fragments allow unobstructed detection of antigens in the 50 kDa or 25 kDa ranges, respectively.
When labeled secondary antibodies specific for both heavy and light chains of IgG, e.g. anti-IgG (H+L), are used to detect protein bands on Western blots following immunoprecipitation (IP), two heavy bands appear (Figure 1 A) 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. However, when labeled anti-IgG, Light Chain Specific antibodies are used for detection, they bind only to the light chain band on the blot (Figure 1 B) and to light chains on the native primary antibodies used for detection. Therefore, a 50 kDa protein may be detected on blots without interference from the heavy chain of the precipitating IgG.
Light chain specific antibodies are available directed against goat, mouse, rabbit, rat and sheep. They have been adsorbed to minimize cross-reactivity with immunoglobulins from many other species, which also may be present on blots.
To detect a 25 kDa analyte without interference from reduced and denatured IgG at 25 kDa, use Anti-Fc specific antibodies, along with a FabuLight™ block.