Rat Pure-Blot anti-Mouse IgG (H+L) Secondary Antibody (eB144) [HRP]

Western Blot: Rat Pure-Blot anti-Mouse IgG (H+L) Secondary Antibody (eB144) [HRP] [NBP3-11661] - Western Blot of Rat Pure-Blot anti-Mouse IgG Secondary antibody (eB144) [HRP]. Lane 1: Mouse IgG, non-denatured. Lane 2: 5 uL Opal Pre-stained Ladder [HRP] at 1:1,000 for 60 min at RT. Block for 30 min at RT. Predicted/Observed size: 160 kDa non-denatured for Mouse IgG.

Western Blot: Rat Pure-Blot anti-Mouse IgG (H+L) Secondary Antibody (eB144) [HRP] [NBP3-11661] - Caspase 7 was immunoprecipitated from 0.5 ml of 1x10e7 Jurkat cells/ml with 5 ug mouse anti-human Caspase 7. Precipitate from 1x10e6 cells was subjected to electrophoresis, transferred to an PVDF membrane, and Western blotted with anti-Caspase 7 using Rat Pure-Blot anti-Mouse IgG Secondary antibody (eB144) [HRP] (Lane 1) or conventional HRP-conjugated anti-mouse antibody (Lane 2) - note the detection of the heavy and light chains of the immunoprecipitating antibody in Lane 2 but not in Lane 1. When Lane 1 is re-immunoblotted using conventional HRP-conjugated anti-mouse polyclonal antibody (Lane 3), the heavy and light chains are now detected, confirming that although the immunoprecipitating heavy and light chains are present, Rat Pure-Blot anti-Mouse IgG Secondary antibody (eB144) [HRP] detects only native antibody and not denatured heavy and light chains.

• Reactivity: Mu
• Applications: WB, ELISA, IP
• Clone: eB144
• Clonality: Monoclonal
• Host: Rat
• Conjugate: HRP

Rat Pure-Blot anti-Mouse IgG (H+L) Secondary Antibody (eB144) [HRP] Summary

• Description: This secondary antibody was prepared from tissue culture supernatant by Protein G affinity chromatography. Assay by immunoelectrophoresis resulted in a single precipitin arc against Anti-Mouse Serum.
  
  Store at -20C. This product is guaranteed for 6 months upon receipt, when handled and stored as instructed.
• Immunogen: Mouse IgG
• Isotype: IgG
• Clonality: Monoclonal
• Host: Rat
• Purity: Protein G purified

Applications/Dilutions

• Dilutions:
  • ELISA
  • Immunoprecipitation
  • Western Blot 1:1000
• Application Notes: This secondary antibody has been tested in ELISA, IP, and Western blot and may also be used for detection in immunoblotting assays that do not employ immunoprecipitation. Mouse IgG Pure-Blot ULTRA is provided as 1000X solution. To achieve best results when detecting mouse IgG1 subtypes, we recommend performing a dot blot or titration to determine the optimal dilution factor for your desired application. 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. In order 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 2.5mls/blot will yield enough reagent for 8 blots. Note that there are three key procedural considerations: 1. Protein A or G beads may be used with the mouse, goat and sheep Pure-Blot secondaries, but not with the rabbit Pure-Blot secondary. Use of protein A or G beads with the rabbit Pure-Blot will result in contaminating bands. 2. Immunoprecipitate should be completely reduced. 3. BLOTTO/Milk should be used as the blocking protein for the immunoblot.
  
  Biochemical Assay, ChIP, ICC/IF

Packaging, Storage & Formulations

• Storage: Store at -20C. Avoid freeze-thaw cycles.
• Buffer: 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: 0.01% Gentamicin Sulfate
• Purity: Protein G purified

Alternate Names for Rat Pure-Blot anti-Mouse IgG (H+L) Secondary Antibody (eB144) [HRP]

• IP Detection Reagent

Background

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

Limitations

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.