An HRP-conjugated anti-mouse IgG (heavy and light chain) secondary antibody was used as the secondary detection reagent for lanes 5 and 6. TidyBlot Western Blot Detection Reagent:HRP was used as the secondary detection reagent for lanes 2 and 3. Lanes 3 and 6 were secondary-antibody-only controls (no primary antibody was added) to determine background staining from the secondary detection reagents. Mouse anti-GSTP1 PrecisionAb™ Antibody was used as a primary antibody for the detection of the GSTP1 protein (~25 kD) in lanes 2 and 5. Lanes 1 and 4 were loaded with Precision Plus Protein™ All Blue Protein Standard. K562 cell lysate (25 µg) spiked with 1 µg of mouse monoclonal antibody of the IgG2b isotype was run in lanes 2, 3, 5, and 6 under reduced conditions for SDS-PAGE and the proteins were transferred onto a nitrocellulose membrane. TidyBlot Western Blot Detection Reagent:HRP binds exclusively to the native anti-GSTP1 antibody, whereas the HRP-conjugated anti-mouse IgG heavy and light chain secondary antibody recognizes both the native anti-GSTP1 and denatured antibodies (as shown by the detection of heavy and light chains) in this mock IP experiment. Here are some fun band detection exercises to challenge your western blot–trained eyes:įig. The result is a cleaner western blot, as the IgG heavy and light chain bands do not show up (see Figures 1, 2, and 3 below). It is HRP conjugated and binds exclusively to the native nondenatured IgG antibody used for western blot detection and thereby binds only to the IgG of interest. The new detection reagent from Bio-Rad, TidyBlot™ Western Blot Detection Reagent, does exactly that. This is a sure way to reduce or eliminate the problem of background band detection in western blots. The best way to avoid this problem is to use a secondary antibody or a detection reagent that binds only to the native nondenatured antibodies rather than to any IgG present in the sample. How do you avoid visualization of IgG heavy or light chains when your immunoprecipitated protein of interest has a molecular weight close to 25 or 50 kD? Bands from IgG heavy (~50 kD) and light (~25 kD) chains often mask or obstruct the bands of interest in a western blot of immunoprecipitated proteins. In addition to the endogenous IgGs, primary antibodies of the IgG isotypes are also released from beads during immunoprecipitation (IP) procedures. In general, they react with both the IgG heavy and light chain.īesides the desired binding to the primary antibody, heavy and light chain–specific secondary antibodies tend to bind to heavy and light chains of endogenous immunoglobulin molecules present in reduced or denatured western blot samples. Secondary antibodies are directed against the IgG class or subclass of the species in which the primary antibody was raised. How many times have we seen bands from IgG heavy or light chains interfering with our bands of interest? Typically the source of this problem is the secondary antibody we use.
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