Thermo Scientific Pierce Poly-HRP Conjugates 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. Pierce Poly-HRP Conjugates 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. Together, these features provide consistent and reliable sensitivity and deliver higher sensitivity than conventional HRP and poly-HRP conjugates without the need for additional signal amplification steps. The Pierce Streptavidin, Goat anti-Mouse and Goat anti-Rabbit Poly-HRP are compatible with chromogenic, fluorogenic and chemiluminescent HRP substrates used in ELISA, Western blotting, immunohistochemistry (IHC) and nucleic acid hybridization assays.

About using Poly-HRP conjugated secondary detection reagents.

We offer several forms of HRP conjugated secondary antibodies and biotin binding proteins that do not contain polymerized HRP (poly-HRP) which are suitable for most immunoassays. These commonly used HRP-conjugates are limited to 1-3 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 than can be achieved with standard HRP conjugates, methods such as peroxidase anti-peroxidase (PAP) systems or avidin-biotin complexes (ABC) may be used. However, these methods increase the cost of the assay and the number of steps that must be optimized. Pierce Poly-HRP Conjugates circumvent such problems because it is comprised of polymers of HRP molecules subsequently conjugated to secondary antibody or streptavidin in a proprietary manufacturing process that increases the molar ratio of HRP on the conjugate while maintaining functionality. The increased molar ratio of HRP molecules per complex is higher, resulting in more HRP molecules being available per binding event to react with the substrate. Therefore, Pierce Poly-HRP Conjugates provided 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.

Highlights for Poly-HRP Conjugates:

• 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 Streptavidin Poly-HRP Conjugate enables superior sensitivity in ELISA applications. Pierce Streptavidin Poly-HRP, a similar product offered by another supplier (Supplier S) and a non-polymerized streptavidin-HRP conjugate (Supplier Z) were compared in an IFNγ ELISA. The Pierce Streptavidin Poly-HRP is significantly more sensitive than the non-polymerized HRP and poly-HRP from Supplier S. This assay was performed with the reagents and protocol supplied with the Thermo Scientific Human IFN gamma Screening Set (Product #ESS0002) by subsituting the 3 different streptavidin-HRP conjugates (50 ng/ml) above for that supplied in the kit and developing ELISA according to the protocol.

 

A.	GAM Poly-HRP	GAM-HRP	Thermo Scientific Pierce Poly-HRP Secondary Antibodies enable low-level target detection in Western blotting applications. Panel A: Blots were probed with 2,000 ng/ml of either mouse anti-AKT 1 antibody or mouse anti-SRC antibody and detected with 80 ng/ml of either goat anti-mouse (GAM) HRP or Pierce Goat Anti-Mouse Poly-HRP. Panel B: Blots were probed with 2,000 ng/ml of either rabbit anti-cytokeratin 18 antibody or rabbit anti-BAX 1 antibody and detected with 80 ng/ml of either goat anti-rabbit (GAR) HRP or Pierce Goat Anti-Rabbit Poly-HRP. Lanes 1–5 contained 25, 12.5, 6.25, 3.1, 1.6 ng of HeLa cell lysate (AKT1, cytokeratin 18 and BAX1 detection) or A549 cell lysate (SRC detection), respectively.
AKT1 SRC
B.	GAR Poly-HRP	GAR-HRP
Cytokeratin 18 BAX1

 

Thermo Scientific Pierce Streptavidin Poly-HRP produces immunohistochemical images with outstanding clarity. Mounted tissues were incubated overnight at 4°C with biotinylated anti-mouse cytokeratin 18 at 18.6 µg/ml. The negative control tissue was incubated with blocking buffer only. Tissues were incubated with 4 µg/ml of either Pierce Streptavidin Poly-HRP or a similar product from Supplier S.

 

Product recommendations based on relative antigen abundance. To aid in choosing an appropriate HRP-conjugate for a specific application, the following table has been provided as a guideline based on the antigen abundance in a typical cell lysate (except as noted). Results may vary with specific cell type, detection system, substrate and assay conditions.
Abundance (Copies per cell)	Target Antigen	Recommended HRP conjugate
Low (10-10,000)	Transcription factors: STAT1, NFκB Cytokines: TNFα, INFγ (in culture media) Low abundant GTPases: GPCP	Pierce Poly-HRP Conjugates
Medium (10,000-100,000)	MAP kinases: Erk1, Erk2 Growth and proliferation kinases: AKT Phosphatases: PP2A Tyrosine kinase receptors: VEGFr, EGFr, PDGFr Cyclin dependent kinases: Cdk2 Cyclins: Cyclin E, Caspase Adaptor proteins: Grb2, Plcγ, SOS	Pierce High Sensitivity HRP Conjugates or if optimzed to lower concentrations: Pierce Poly-HRP Conjugates
High (>100,000)	Cytoskeletal protein: Actin, myosin, tubulin, vimentin Chaperon proteins: HSP70, HSP90 Histones: H2A, H4 Metabolic enzymes: GAPDH, Cyclophilin A, Cyclophilin B	Standard HRP Conjugates or if optimzied to lower concentrations: Pierce High Sensitivity HRP Conjugates

 

Recommended HRP-conjugate concentrations by application. The following guidelines are provided as a starting point for optimizing HRP-conjugate concentration in ELISA, Western and immunohistochemistry applications.
	ELISA and Westerns	Immunohistochemistry
Standard HRP Conjugates	100 – 200 ng/ml	5 – 100 µg/ml
Pierce High Sensitivity HRP Conjugates	50 – 100 ng/ml	2 – 4 µg/ml
Pierce Poly-HRP Conjugates	50 – 100 ng/ml	2 – 4 µg/ml

Related Products:
Secondary Antibodies and Conjugates – find other conjugated secondary antibodies and biotin binding proteins
High Sensitivity HRP Conjugates – high performance HRP conjugates for routine applications
Western Blotting, ELISA and Cell Imaging Reagents
Blocking Buffers
Secondary Antibody Selection Guide

Product #	Description	Pkg. Size
21140	Pierce Streptavidin Poly-HRP Concentration: 0.5 mg/ml	0.5 ml
32230	Pierce Goat Anti-Mouse Poly-HRP Concentration: 0.5 mg/ml	0.5 ml
32260	Pierce Goat Anti-Rabbit Poly-HRP Concentration: 0.5 mg/ml	0.5 ml