Rabbit anti-Human IgG (H+L) Secondary Antibody [Biotin]

Western Blot: Rabbit anti-Human IgG (H+L) Secondary Antibody [Biotin] [NB120-6758] - Lane 1: Human IgG Load: 50 ng per lane Primary antibody: Human IgG (H&L) Antibody Biotin Conjugated at 1:1000 for 60 min RT Secondary antibody: HRP Conjugated Streptavidin at 1:40,000 for 30 min at RT Block: incubated with blocking buffer for 30 min at RT

• Reactivity: Hu
• Applications: WB, ELISA, IHC
• Clonality: Polyclonal
• Host: Rabbit
• Conjugate: Biotin

Rabbit anti-Human IgG (H+L) Secondary Antibody [Biotin] Summary

• Description: For extended storage aliquot contents and freeze at -20C or below. Avoid cycles of freezing and thawing. Centrifuge product if not completely clear after standing at room
  
  This product was prepared from monospecific antiserum by immunoaffinity chromatography using Human IgG coupled to agarose beads followed by solid phase adsorption(s) to remove any unwanted reactivities. Assay by immunoelectrophoresis resulted in a single precipitin arc against anti-biotin, anti-Rabbit Serum, Human IgG and Human Serum.
• Immunogen: Human IgG whole molecule
• Isotype: IgG
• Clonality: Polyclonal
• Host: Rabbit
• Purity: Multi-step

Applications/Dilutions

• Dilutions:
  • ELISA 1:20000 - 1:100000
  • Immunohistochemistry 1:1000 - 1:5000
  • Peptide ELISA
  • Western Blot 1:2000 - 1:10000
• Application Notes: This product has been tested by western blot and ELISA and has been assayed against 1.0 ug of Human IgG in a standard capture ELISA using Peroxidase Conjugated Streptavidin and ABTS (2,2'-azino-bis-[3-ethylbenthiazoline-6-sulfonic acid]) as a substrate for 30 minutes at room temperature. A working dilution of 1:50,000 to 1:220,000 of the reconstitution concentration is suggested for this product.
  
  Use in Peptide ELISA was reported in the scientific literature (PMID: 22720696).
• Readout System:
  • Streptavidin Native Protein NBP2-29367
  • Streptavidin Native Protein NBP2-29370
  • Streptavidin Native Protein NBP2-29371

Packaging, Storage & Formulations

• Storage: Store at 4C short term. Aliquot and store at -20C long term. Avoid freeze-thaw cycles.
• Buffer: 0.02 M Potassium Phosphate, 0.15 M Sodium Chloride, pH 7.2, 10 mg/mL Bovine Serum Albumin (BSA) - Immunoglobulin and Protease free
• Preservative: 0.01% Sodium Azide
• Purity: Multi-step

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.

Publications for IgG (H+L) Antibody (NB120-6758)(1)

Publication using NB120-6758

• Kukreja M, Johnston SA, Stafford P. Comparative study of classification algorithms for immunosignaturing data BMC Bioinformatics 2012-06-23 [PMID: 22720696] (PEP-ELISA)