Thermo Scientific Pierce Poly-HRP Secondary Antibodies are goat anti-mouse IgG and goat anti-rabbit IgG polyclonal antibodies that are conjugated with polymers of horseradish peroxidase, enabling one-step signal amplification and detection of primary antibodies for IHC and other methods.
These poly-HRP antibodies are designed to deliver the highest sensitivity and low background in immunoassays where sample volume is limited or when the target molecule is present at low levels. The secondary antibodies are purified to remove unconjugated probe molecules that reduce signal intensity by competing for binding sites with horseradish peroxidase (HRP)-conjugated molecules. In addition, these conjugates are free of HRP monomers that may lead to increased background signal. Both Pierce Goat Anti-Mouse (GAM) and Goat Anti-Rabbit (GAR) Poly-HRP Antibodies are compatible with chromogenic, fluorogenic and chemiluminescent HRP substrates used in ELISA, Western blotting, immunohistochemistry (IHC) and nucleic acid hybridization assays.
- High sensitivity – detect low-abundant targets (low picogram-femtogram range) with high signal-to-noise ratios
- Robust – regent is free of monomer conjugates and unconjugated molecules for highest specific activity and minimal background
- Flexible – compatible with chromogenic, fluorogenic and chemiluminescent substrates
- Stable – ready-to-use stabilized liquid format stored at 4°C
- Cost effective – use less conjugate per assay
- Versatile – compatible with ELISA, Western blotting and immunohistochemistry
- Easy to use – can be directly substituted into immunoassays and other detection assays
Thermo Scientific Pierce Poly-HRP Secondary Antibodies enable low-level target detection in Western blotting applications.
Panel A: Blots were probed with 2µg/mL of either mouse anti-AKT1 antibody or mouse anti-SRC antibody and detected with 80ng/mL of either goat anti-mouse (GAM) HRP or Pierce Goat Anti-Mouse Poly-HRP.
Panel B: Blots were probed with 2µg/mL of either rabbit anti-cytokeratin 18 antibody or rabbit anti-BAX1 antibody and detected with 80ng/mL of either goat anti-rabbit (GAR) HRP or Pierce Goat Anti-Rabbit Poly-HRP.
Lanes 1 to 5 contained 25, 12.5, 6.25, 3.1, 1.6ng of HeLa cell lysate (AKT1, cytokeratin 18 and BAX1 detection) or A549 cell lysate (SRC detection), respectively.
Several forms of HRP conjugated secondary antibodies are available that do not contain polymerized HRP (poly-HRP) and are suitable for typical immunoassays. These and other commonly used HRP-conjugated antibodies and biotin-binding proteins are limited to one to three HRP molecules per protein in order to maintain functionality of the conjugate. This molar ratio of conjugation is effective in routine experiments but may lack the sensitivity required for detecting targets present in low picogram to femtogram amounts without the use of additional steps to amplify the signal.
When a higher degree of signal amplification is needed with standard HRP conjugates, methods such as peroxidase anti-peroxidase (PAP) systems or avidin-biotin complexes (ABC) can be used. However, these methods increase the cost of the assay and the number of steps that must be optimized. Pierce Poly-HRP Secondary Antibodies circumvent such problems because they use polymers of HRP molecules that are conjugated in a proprietary manufacturing process that increases the molar ratio of HRP on the conjugates without compromising antibody function. The increased molar ratio of HRP molecules to antibody results in more HRP molecules being available per binding event to react with the substrate. Therefore, Pierce Poly-HRP Conjugates provide substantial signal amplification over other HRP-conjugates, eliminating the need for additional steps and saving time and expense.
As with all immunoassays, optimization is required to achieve the best signal to noise ratio. Switching to higher sensitivity substrates and conjugates can reveal more desired signal as well as background signal. If this occurs, follow standard primary and secondary dilution techniques or perform blocking buffer optimization. See Streptavidin Poly-HRP Conjugate for recommendations about choosing an appropriate type of HRP conjugate.
Protein Methods Library: Overview of Immunohistochemistry
All Secondary Antibodies and Conjugates
Standard Anti-Mouse IgG Antibodies
Standard Anti-Rabbit IgG Antibodies
Streptavidin Poly-HRP Conjugate
ABC Staining Kits