IMMUNOPRECIPITATION WESTERN BLOT PLUS

What do the research community say about TidyBlot?īio-Rad customers are successfully using TidyBlot to obtain high quality, publishable western blotting data. Please note that TidyBlot is HRP conjugated and that the western blot bands (red) and protein standards (green) have been pseudocolored. Arrow points to GAPDH (molecular weight 37 kDa). HRP conjugated Tidyblot ( STAR209P) was used at 1/200 and visualized on the ChemiDoc™ MP.

IMMUNOPRECIPITATION WESTERN BLOT PLUS

8.75 µl Jurkat whole cell lysate (WCL) was run in lanes 5 & 11 while Precision Plus Protein™ Prestained Standards were run in lane 6.ġb) Rabbit anti-GAPDH antibody ( VPA00187) was used at 1/1000 in lanes 1-5. IP was performed on Jurkat cell lysates using 10 µg (lanes 1 & 7), 5 µg (lanes 2 & 8), 1 µg (lanes 3 & 9) mouse anti-GAPDH antibody ( MCA4739) and 10 µg mouse IgG1 negative control ( MCA1209) (lanes 4 & 10).ġa) 8.75 µl of each IP was loaded onto an AnykD™ Criterion™ TGX Stain-Free™ gel. Western blot analysis of GAPDH IP samples. TidyBlot, in contrast to conventional secondary antibodies, only binds to the native antibody used during the western blotting procedure and therefore enables the western blot detection of IP samples without interference from IgG heavy and light chains. The animation below outlines the different steps in an IP procedure using SureBeads Magnetic Beads and highlights how you can use TidyBlot to mitigate obstruction from IgG heavy and light chains. TidyBlot Reagent prevents detection of endogenous immunoglobulins The IgG chains might mask the protein of interest and make detection difficult especially when the immunoprecipitated proteins have a molecular weight of ~50 kDa or 25 kDa. This binding results in two distinct bands that can be observed on the western blot the IgG heavy chain at 50 kDa and the IgG light chain at 25 kDa. These IgGs then become denatured during the IP sample preparation procedure and are detected by conventional heavy and light chain secondary antibodies used in western blotting experiments. Western blot detection of immunoprecipitated proteins is a commonly used technique to study protein-protein interactions and immunoprecipitation (IP) is. In contrast to other sample types, the observed western blot background when detecting IP samples is often higher due to antibodies/IgGs being released from beads during the IP procedure. Western blot detection of immunoprecipitated proteins is a commonly used technique to study protein-protein interactions and immunoprecipitation (IP) is often performed to enrich low abundant proteins in a sample to enable their detection.

Immunoprecipitation western blot