Goat anti-Rat IgG (H+L) Secondary Antibody (Pre-adsorbed)

Western Blot: Goat anti-Rat IgG (H+L) Secondary Antibody (Pre-adsorbed) [NB120-7092] - Western Blot of Goat anti-Rat IgG (H+L) Secondary antibody (Pre-adsorbed). Lane M: 3 ul Molecular Ladder. Lane 1: Rat IgG whole molecule. Lane 2: Rat IgG F(c) Fragment. Lane 3: Rat IgG Fab Fragment. Lane 4: Rat IgM Whole Molecule. Lane 5: Rat Serum. All samples were reduced. Load: 50 ng per lane. Block for 30 min at RT. Primary Antibody: Goat anti-Rat IgG (H+L) Secondary antibody (Pre-adsorbed) 1:1,000 for 60 min at RT. Secondary Antibody: Anti-Goat IgG (DONKEY) Peroxidase Conjugated Antibody 1:40,000 in blocking buffer for 30 min at RT. Predicted/Obsevered Size: 25 and 55 kDa for Rat IgG and Serum, 25 kDa for F(c) and Fab, 78 and 25 kDa for IgM. Rat F(c) migrates slightly higher.

Western Blot: Goat anti-Rat IgG (H+L) Secondary Antibody (Pre-adsorbed) [NB120-7092] - tubulin was detected using a Dye Light 680 conjugate. Anti-TNFa was detected using a Dye Light 800 conjugate.

Western Blot: Goat anti-Rat IgG (H+L) Secondary Antibody (Pre-adsorbed) [NB120-7092] - Western blot of CY3 Conjugated Goat Anti-Rat IgG Pre-Adsorbed secondary antibody. Lane 1: Rat IgG. Lane 2: None. Load: 50 ng per lane.Primary antibody: None.Secondary antibody: CY3 goat secondary antibody at 1:1,000 for 60 min at RT. Blocking: for 30 min at RT.Predicted/Observed size: 25,55 kDa, 25, 55 kDa for Rat IgG.Other band(s): Rat IgG splice variants and isoforms.

Dot Blot: Goat anti-Rat IgG (H+L) Secondary Antibody (Pre-adsorbed) [NB120-7092] - Dot Blot of Goat anti-Rat IgG (H+L) Secondary Antibody [Alkaline Phosphatase] (Pre-adsorbed).Antigen: Rat IgGLoad: 1 ugPrimary antibody: Goat anti-Rat IgG (H+L) Secondary Antibody [Alkaline Phosphatase] (Pre-adsorbed) 1:1000 in Fluorescent blocking buffer for 60 min at RT.Alkaline Phosphatase Substrate Neat for 30 seconds at RT.Block: Fluorescent blocking buffer for 60 min at RT.. Using the Alkaline Phosphatase format of this antibody.

Western Blot of Goat anti-Rat IgG (H+L) Secondary Antibody (Pre-adsorbed)

• Reactivity: Rt
• Applications: WB, DB, ELISA, Flow, IHC
• Clonality: Polyclonal
• Host: Goat
• Conjugate: Unconjugated
• Format: Pre-adsorbed

Goat anti-Rat IgG (H+L) Secondary Antibody (Pre-adsorbed) Summary

• Description: This product was prepared from monospecific antiserum by immunoaffinity chromatography using Rat 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-Goat Serum, Rat IgG and Rat Serum
  
  Store vial at 4C prior to opening. This product is stable for several weeks at 4C as an undiluted liquid. Dilute only prior to immediate use. For extended storage aliquot contents and freeze at -20C or below. Avoid cycles of freezing and thawing.
• Immunogen: Rat IgG whole molecule
• Specificity: This antibody was pre-adsorbed against Bovine, Chicken, Goat, Guinea Pig, Hamster, Horse, Human, Mouse, Rabbit, and Sheep Serum Proteins.
  
  No reaction was observed against Bovine, Chicken, Goat, Guinea Pig, Hamster, Horse, Human, Mouse, Rabbit and Sheep Serum Proteins.
• Isotype: IgG
• Clonality: Polyclonal
• Host: Goat
• Purity: Multi-step

Applications/Dilutions

• Dilutions:
  • Dot Blot
  • ELISA 1:10000 - 1:40000
  • Flow Cytometry
  • Immunohistochemistry 1:500 - 1:2000
  • Western Blot 1:1000 - 1:4000
• Application Notes: This product has been tested by ELISA and western blot and is suitable for ELISA, western blot, and immunohistochemistry, as well as other assays requiring lot-to-lot consistency. Specific conditions should be optimized by user.
  
  Use in Flow Cytometry was reported in the scientific literature (PMID: 2841832).

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
• 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 (NB120-7092)(1)

Publication using NB120-7092

• Park CG, Rodriguez A, Steinman RM. PE-Cy5.5 conjugates bind to the cells expressing mouse DEC205/CD205 J Immunol Methods 2012-07-31 [PMID: 22841832] (FLOW)