"I have used a wide variety of secondaries and Jackson ImmunoResearch has consistently been the best. The fluorophores are bright and stable and their selective (x reactivity removed) secondaries have always shown species specificity in multiple labeling."
Horseradish peroxidase crystal structure at 1.57 Å from PDB 1HCH. Images generated using Molsoft ICM browser. Image shows two calcium molecules (grey spheres) and the catalytic iron (orange sphere) within the protoporphyrin IX container.
This commonly used reporter enzyme is derived from the root of the horseradish plant (Armoracia rusticana). JIR HRP conjugates are prepared by a modified Nakane and Kawaoi procedure (1974).
HRP conjugates are suitable for all immunotechniques employing colorimetric and chemiluminescent detection methods, including Western blotting, immunohistochemistry and ELISA. Jackson ImmunoResearch provides a wide range of secondary antibodies, with comprehensive options for host and target species, as well as immunoglobulin specificities.
Horseradish peroxidase is available conjugated to:
Some tissues contain endogenous peroxidase-like enzymes which can react with peroxidase substrates, resulting in background staining. Pre-treatment of sample with hydrogen peroxidase will exhaust the endogenous enzyme activity, allowing clear detection of specific signal.
If glycerol is added to extend shelf life of reconstituted product, confirm that glycerol is ACS grade or better. Lower grades of glycerol may seriously inhibit peroxidase enzyme activity.
Colorimetric detection
HRP conjugates can be used for colorimetric detection. The conjugated reporter enzyme catalyzes the conversion of the chromogenic substrate to a colored precipitate, visualized directly on the blotting membrane or tissue sample. Colorimetric detection can offer quick and easily obtained results without the need for expensive detectors or extensive optimization.
Enzyme-linked conjugates can also be used for chemiluminescent signal detection. HRP conjugates produce signal by oxidizing a chemiluminescent substrate (luminol) to a form which emits light. AP conjugates produce signal when the enzyme dephosphorylates a specific substrate (e.g. 1,2-dioxetane) to a light emitting product. The signal can then be captured by exposing photographic film to the membrane or using a cooled charge-coupled device (CCD) camera. Chemiluminescent detection offers excellent sensitivity, however quantification and probing for multiple targets can be limited, and development may require refinement to optimize signal capture.
A colorimetric Western blot. Heavy (HC 50 kDa) and light (LC 25 kDa) chains of reduced and SDS-denatured mouse IgG separated by SDS-PAGE and detected by Western blot using Peroxidase-Goat anti-Mouse IgG (H+L) (115-035-003) and visualized with TMB chromogenic substrate.
Bovine IgG and Fc fragment specific
Chicken IgY (IgG), F(ab')2 and Fc fragment specific
Cat IgG, F(ab')2 and Fc Fragment specific
Dog IgG and F(ab')2 fragment specific
Goat IgG, F(ab')2, Fc fragment, and light chain specific
Guinea Pig IgG, F(ab')2 and Fc specific
Armenian Hamster IgG
Syrian Hamster IgG
Horse IgG and Fc fragment specific
Human IgA + IgG + IgM
Human Serum IgA, α Chain Specific
Human IgG, F(ab')2, Fcγ Fragment and light chain specific
Human IgM, Fc5μ fragment specific
Human Lactoferrin
Mouse IgG, Fcγ fragment specific
Mouse IgG, Fcγ subclass specific (1 / 2a / 2b / or 3)
Mouse IgM and µ chain specific
Rabbit IgG, F(ab')2, Fc and light chain specific
Rat IgG, Fcγ Fragment specific
Rat IgM and IgM μ chain specific
Sheep IgG, F(ab')2, Fc fragment and light chain specific
Swine IgG
anti-biotin
anti-digoxin
anti-fluorescein
Anti-Horseradish Peroxidase Antibodies
Affinity-purified anti-horseradish peroxidase antibodies are available for detection of horseradish peroxidase antigen, or for signal amplification of HRP-containing reagents. For immunostaining of mammalian cells, an advantage of using anti-horseradish peroxidase is reduced background, since the antibody does not recognize the endogenous peroxidase-like enzymes found in those cells.
Paul K. Nakane, Akira Kawaoi (1974). Peroxidase-labeled Antibody A New Method Of Conjugation Journal of Histochemistry & Cytochemistry Vol 22, Issue 12, pp. 1084 - 1091. 10.1177/22.12.1084
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