Western Blot Detection of IP Samples

Western Blot Detection of IP Samples

using TidyBlot™

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.

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. 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. 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. 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.

The below workflow 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, 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. 

Overview of actin gamma IP procedure using SureBeads Magnetic Beads followed by western blot detection of the IP sample with an anti-actin gamma primary antibody and a standard secondary antibody or TidyBlot.
 

Figure 1: Overview of actin gamma IP procedure using SureBeads Magnetic Beads followed by western blot detection of the IP sample with an anti-actin gamma primary antibody and a standard secondary antibody or TidyBlot. Using TidyBlot enables the detection of actin gamma without interference from the IgG heavy chain; the molecular weights of actin-gamma (46 kDa) and the IgG heavy chain (50 kDa) are very close, which often results in masking of the actin gamma band by the IgG heavy chain.

Example of an IP experiment using TidyBlot as the western blot detection reagent:

Figure 2: Western blot analysis of GAPDH IP samples. 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).

1a) 8.75 µl of each IP was loaded onto an AnykD™ Criterion™ TGX Stain-Free™ gel. 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.

1b) Rabbit anti-GAPDH antibody (VPA00187) was used at 1/1000 in lanes 1-5. HRP conjugated Tidyblot (STAR209P) was used at 1/200 and visualized on the ChemiDoc™ MP. Arrow points to GAPDH (molecular weight 37 kDa). Please note that TidyBlot is HRP conjugated and that the western blot bands (red) and protein standards (green) have been pseudocolored.