Pig anti-Mouse IgG (H+L) Secondary Antibody [FITC]

Immunocytochemistry/Immunofluorescence: Pig anti-Mouse IgG (H+L) Secondary Antibody [FITC] (Azide Free) [NBP2-69411] - Pig anti-Mouse IgG (H+L) Secondary Antibody [FITC] (Azide Free) was used in a 1:25 dilution on swine colon, in combination with the primary monoclonal antibody to vimentin, RV202, used in a 1:1000 dilution. The green staining shows the connective tissue cells, while no staining is seen in the epithelial cells of the intestinal crypts.

• Reactivity: Mu
• Applications: ELISA, ICC/IF, IHC
• Clonality: Polyclonal
• Host: Pig
• Conjugate: FITC
• Concentration: LYOPH

Pig anti-Mouse IgG (H+L) Secondary Antibody [FITC] Summary

• Description: The lyophilized conjugate is shipped at ambient temperature and may be stored at 4C; prolonged storage at or below -20C. Prior to use, an aliquot is thawed slowly in the dark at ambient temperature, spun down again and used to prepare working dilutions by adding sterile phosphate buffered saline (PBS, pH 7.2). Repeated thawing and freezing should be avoided. Working dilutions should be stored at 4C, not refrozen, and preferably used the same day. If a slight precipitation occurs upon storage, this should be removed by centrifugation. It will not affect the performance of the immunoconjugate.
• Immunogen: Purified normal IgG isolated from pooled mouse serum. Freund's complete adjuvant is used in the first step of the immunization procedure.
• Specificity: The reactivity of the antiserum is directed to the Fc and Fab subunits of the IgG molecule. It includes a certain degree of reactivity with other immunoglobulins via the common Fab portion. It does not react with any non-Ig protein in mouse serum, as tested by immunoelectrophoresis and double radial immunodiffusion.
• Clonality: Polyclonal
• Host: Pig
• Purity: Protein G purified

Applications/Dilutions

• Dilutions:
  • ELISA 1:100-1:2000
  • Immunocytochemistry/ Immunofluorescence 1:10 - 1:500
  • Immunohistochemistry
  • Immunohistochemistry-Frozen
  • Immunohistochemistry-Paraffin 1:10 - 1:500
• Application Notes: (In)direct immunofluorescence

Reactivity Notes

Inter-species cross-reactivity is a normal feature of antibodies to immunoglobulins, since Ig of different species frequently share antigenic determinants. Cross-reactivity of this antiserum has not been tested in detail.

Packaging, Storage & Formulations

• Storage: Store at 4C in the dark.
• Buffer: Lyophilized from PBS (pH 7.2)
• Preservative: No Preservative
• Concentration: LYOPH
• Purity: Protein G purified
• Reconstitution Instructions: Reconstitute by by adding 2 ml sterile distilled water, spun down to remove insoluble particles, divided into small aliquots, frozen and stored at or below -20C.

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.