Mouse Pure-Blot anti-Rabbit IgG (H+L) Secondary Antibody (eB182) [HRP]

Novus Biologicals | Catalog # NBP3-11668

Novus Biologicals

Key Product Details

Species Reactivity

Rabbit

Applications

Western Blot, ELISA, Immunoprecipitation

Label

HRP

Antibody Source

Monoclonal Mouse IgG Clone # eB182
View all IgG (H+L) Secondary Antibodies »

Product Specifications

Immunogen

Rabbit IgG

Clonality

Monoclonal

Host

Mouse

Isotype

IgG

Description

This secondary antibodye was prepared from tissue culture supernatant by Protein G affinity chromatography. Assay by immunoelectrophoresis resulted in a single precipitin arc against Anti-Rabbit Serum.

Store at -20C. This product is guaranteed for 6 months upon receipt, when handled and stored as instructed.

Scientific Data Images

Western Blot: Mouse Pure-Blot anti-Rabbit IgG (H+L) Secondary Antibody (eB182) [HRP] [NBP3-11668] - Jurkat cell lysate (0.5 ml of 1x10e7 cells/ml) was incubated with rabbit anti-human Stat1 and immunoprecipitated using Protein G, Protein A and Anti-Rabbit Ig IP Beads. Precipitate from 5x10e5 cells was subjected to electrophoresis, transferred to a PVDF membrane, and Western blotted with anti-Stat1 using Mouse Pure-Blot anti-Rabbit IgG Secondary antibody (eB182) [HRP].

Applications

Application
Recommended Usage

Western Blot

1:1000
Application Notes
This secondary antibody has been tested in ELISA, Western blot, and immunoprecipitation and may also be used for detection in immunoblotting assays that do not employ immunoprecipitation. Rabbit IgG Pure-Blot is provided as 1000X solution. To conserve reagent, we recommend incubating the blots from minigels in sealed bags (removing as much air as possible) with minimal volume (2-3 mLs). If used conservatively at 2.5 mLs/blot will yield enough reagent for 20 blots. Note that there are three key procedural considerations: 1. Protein A or G should not be used for the immunoprecipitation. Use of protein A or G beads with the rabbit Pure-Blot will result in contaminating bands. For immunoprecipitation, Anti-Rat IgG beads, or Anti-Rabbit IgG beads should be used for rat or rabbit immunoprecipitating antibodies, respectively. 2. Immunoprecipitate should be completely reduced. 3. BLOTTO/Milk should be used as the blocking protein for the immunoblot. All recommended dilutions for listed applications are intended as an initial recommendation, specific conditions for each protein and antibody combination should be specifically optimized by the end user.

ChIP, FISH, IF

Formulation, Preparation, and Storage

Purification

Protein G purified

Formulation

0.02 M Potassium Phosphate, 0.15 M Sodium Chloride, pH 7.2, 0.1 mg/ml Bovine Serum Albumin (BSA, IgG and Protease free), 50% (v/v) Glycerol

Preservative

ProClin 150

Concentration

Please see the vial label for concentration. If unlisted please contact technical services.

Shipping

The product is shipped with polar packs. Upon receipt, store it immediately at the temperature recommended below.

Stability & Storage

Store at -20C. Avoid freeze-thaw cycles.

Background: IgG (H+L)

Antibodies, also known as immunoglobulins (Igs) are critical for immunity and are grouped into five primary classes: IgG, IgM, IgA, IgD, and IgE. The most abundant antibody isotype is immunoglobulin G (IgG) with concentrations ranging from 7.5-22 mg/ml in human serum and has a molecular weight of 150 kDa. The major effector functions of IgG include neutralization, opsonization, complement fixation and antibody dependent cell-mediated cytotoxicity (ADCC). This monomeric immunoglobulin, expressed on the surface of mature B cells, is often depicted as a Y-shape and comprised of 2 heavy chains and 2 light chains linked by disulfide bonds. The heavy chain is type gamma including subtypes gamma 1, gamma 2, gamma 3, and gamma 4 while the light chain is either a kappa or lambda chain. An IgG molecule has two antigen binding sites, each consisting of a heavy and light chain N-terminal variable domain. When combined with the constant heavy chain 1 (Ch1) and the constant light chain domains, it forms the fragment antigen-binding (Fab) region (2 per antibody). The remaining domains (Ch2-Ch4) of both heavy chains make up the Fc region and contain a site for covalently linking an enzymatic or fluorochrome probe, such as HRP or Janelia Fluor 549, for target detection and visualization (1,2,3).

The 4 IgG subclasses, sharing 95% amino acid identity, include IgG1, IgG2, IgG3, and IgG4 for humans and IgG1, IgG2a, IgG2b, and IgG3 for mice. The relative abundance of each human subclass is 60% for IgG1, 32% for IgG2, 4% for IgG3, and 4% for IgG4. In an IgG deficiency, there may be a shortage of one or more subclasses (4).

References

1. Painter RH. (1998) Encyclopedia of Immunology (Second Edition). Elsevier. 1208-1211

2. Chapter 9 - Antibodies. (2012) Immunology for Pharmacy. Mosby 70-78

3. Schroeder H, Cavacini, L. (2010) Structure and Function of Immunoglobulins. J Allergy Clin Immunol. 125(2 0 2): S41-S52. PMID: 20176268

4. Vidarsson G, Dekkers G, Rispens T. (2014) IgG subclasses and allotypes: from structure to effector functions. Front Immunol. 5:520. PMID: 25368619

Alternate Names

IP Detection Reagent

Additional IgG (H+L) Products

Product Documents

Certificate of Analysis

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Product Specific Notices

This product is for research use only and is not approved for use in humans or in clinical diagnosis. Secondary Antibodies are guaranteed for 1 year from date of receipt.

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