|Product Name||Rabbit Anti-Human IgG（H+L） Secondary Antibody, HRP Conjugate|
|Synonyms||HRP-conjugated rabbit anti-human IgG|
|Description||Rabbit Anti-Human IgG Secondary Antibody, HRP Conjugate, for the indirect sensitive immunodetection and/or quantification of target proteins through Dotblot, WB, or ELISA by assaying an HRP-catalyzed reaction product in the vicinity of the antigen-primary antibody-secondary antibody-HRP complex.|
|Reagent Type||Secondary antibody, reporter enzyme labeled|
|Label||HRP (Horseradish Peroxidase)|
|Immunogen||Whole molecule human IgG|
|Preparation||Affinity purified from rabbit antiserum|
|Specificyity||Human IgG specific; No cross-reactivity with rat/mouse/goat/rabbit IgG|
|Form Supplied||Liquid: concentrated buffered stock solution|
|Formulation||0.5 mg HRP-conjugated secondary antibody |
0.01 M PBS (pH 7.4)
|Storage||4C for 1 year|
|Precautions||FOR RESEARCH USE ONLY. NOT FOR DIAGNOSTIC OR CLINICAL USE|
|Assay Type||SDS-PAGE separated-, membrane-immobilized-, human primary-antibody-probed proteins from cell/tissue lysates|
|Assay Purpose||Protein detection|
|Technique||Immunodetection of target antibody with HRP reporter enzyme|
|Equipment Needed||WB/Dot blot/ELISA instrumentation; X-ray film cassette or a charge-coupled device (CCD) imager; Spectrophotometer|
|Compatibility with Reagents||Incompatible with sodium azide and metals |
incompatible with high phosphate concentrations
|Specific||High signal-to-noise ratio|
|Sensitive||Detects low-abundant targets due to an optimal number of HRP molecules per antibody|
|High Signal Amplification||Multiple secondary antibodies can bind to a single primary antibody;Secondary antibodies Fc regions provide further binding locations for biotin, or enable the use of ABC and SABC|
|Fast||Generates strong signals in a relatively short time span|
|Quantifieable||Allows quantification of detected signal|
|Easy to Use||Supplied in a workable liquid format|
|Flexible||HRP: compatible with chromogenic, fluorogenic and chemiluminescent substrates;|
|Convenient||HRP’s small size: no interference with the primary/secondary antibody interaction; no steric hindrance to antibody/antigen complexes|
Most commonly, secondary antibodies are generated by immunizing the host animal with a pooled population of immunoglobulins from the target species. The host antiserum is then purified through immunoaffinity chromatography to remove all host serum proteins, except the specific antibody of interest. Purified secondary antibodies are further solid phase adsorbed with other species serum proteins to minimize cross-reactivity in tissue or cell preparations, and are then modified with antibody fragmentation, label conjugation, etc., to generate highly specific reagents. Secondary antibodies can be conjugated to a large number of labels, including enzymes, biotin, and fluorescent dyes/proteins. Here, the antibody provides the specificity to locate the protein of interest, and the label generates a detectable signal. The label of choice depends upon the experimental application.
Horseradish peroxidase (HRP) is extensively used for labeling secondary antibodies in ELISA, western blot, dot blot and immunohistochemistry. The HRP enzyme is made visible using a substrate that, when oxidized by HRP in the presence of hydrogen peroxide as an oxidizing agent, yields a characteristic change that is detectable by specific detection methods. The substrates commonly used with HRP fall into different categories including chromogenic, fluorogenic, and chemiluminescent substrates depending on whether they produce a colored, fluorimetric or luminescent derivative respectively. The intensity of the signal is proportional to peroxidase activity and is a measure of the number of enzyme molecules reacting, hence of the amount of recognized primary antibodies, and thus of the amount of target antigen.
Western Blotting: 1:500-3000(DAB detection)
Western Blotting: 1:3000-10000(Enhanced chemiluminescent detection )
Optimal working dilutions must be determined by end user.
It is not recommended for IHC/ICC.