Donkey anti-Rabbit IgG (H+L) Secondary Antibody (Pre-adsorbed)

Western Blot: Donkey anti-Rabbit IgG (H+L) Secondary Antibody (Pre-adsorbed) [NBP1-72741] - Lane 1: Rabbit IgG Load: 50 ug per lane Secondary antibody: Rabbit IgG (H&L) Antibody DyLight (TM) 488 conjugated Pre-Adsorbed at 1:1,000 for 60 min at RT Block for30 min at RT Predicted/Observed size: 55 and 28 kDa, 55 and 28 kDa Image from the DyLight 488 version of this antibody.

Immunocytochemistry/Immunofluorescence: Donkey anti-Rabbit IgG (H+L) Secondary Antibody (Pre-adsorbed) [NBP1-72741] - Immunocytochemistry/Immunofluorescence: Donkey anti-Rabbit IgG (H+L) Secondary Antibody [DyLight 488] (Pre-absorbed) [NBP1-72966] Image from the DyLight 488 version of this antibody.

Western Blot: Donkey anti-Rabbit IgG (H+L) Secondary Antibody (Pre-adsorbed) [NBP1-72741] - Donkey anti-Rabbit IgG (H+L) Secondary Antibody [DyLight 800] [NBP1-72970] - DyLight (R) dyes can be used for two-color Western Blot detection with low background and high signal

Western Blot: Donkey anti-Rabbit IgG (H+L) Secondary Antibody (Pre-adsorbed) [NBP1-72741] - Lane 1: Rabbit IgG whole molecule. Lane 2: Rabbit IgG F(ab) Fragment. Lane 3: Rabbit IgG F(c) Fragment. Lane 4: Rabbit IgM Whole Molecule. Lane 5: Normal Rabbit Serum. All samples were reduced. Load: 50 ng of IgG, F(ab), F(c) and Serum, 25 ng of IgM. Block: incubated with blocking buffer for 30 min at RT. Primary Antibody: Anti-Rabbit IgG (H&L) (DONKEY) Antibody (Min X Bv Ch Gt GP Ham Hs Hu Ms Rt & Sh Serum Proteins) 1:7,500 for 60 min at RT. Secondary antibody: Anti-Donkey IgG (GOAT) Peroxidase Conjugated Antibody 1:40,000 in incubated with blocking buffer for 30 min at RT. Predicted/Observed Size: 25 and 50 kDa for Rabbit IgG and Serum, 25 kDa for F(c) and F(ab), 70 and 23 kDa for IgM. Rabbit F(c) migrates slightly higher.

Fluorophore-linked immunosorbent assay: Donkey anti-Rabbit IgG (H+L) Secondary Antibody (Pre-adsorbed) [NBP1-72741] - Donkey anti-Rabbit IgG (H+L) Secondary Antibody [DyLight 800] [NBP1-72970]

• Reactivity: Rb
• Applications: WB, ELISA, ICC/IF, IHC
• Clonality: Polyclonal
• Host: Donkey
• Conjugate: Unconjugated
• Format: Pre-adsorbed

Donkey anti-Rabbit IgG (H+L) Secondary Antibody (Pre-adsorbed) Summary

• Description: This product was prepared from monospecific antiserum by immunoaffinity chromatography using Rabbit 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-Donkey Serum, Rabbit IgG and Rabbit 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: Donkey anti-Rabbit IgG (H+L) Secondary Antibody (Pre-adsorbed) was produced by repeated immunization with rabbit whole IgG molecule in donkey.
• Specificity: This antibody was pre-adsorbed against Bovine, Chicken, Goat, Guinea Pig, Hamster, Horse, Human, Mouse, Rat, and Sheep Serum Proteins.
  
  No reaction (<1%) was observed against Bovine, Chicken, Goat, Guinea Pig, Hamster, Horse, Human, Mouse, Rat and Sheep Serum Proteins.
• Isotype: IgG
• Clonality: Polyclonal
• Host: Donkey
• Purity: Multi-step

Applications/Dilutions

• Dilutions:
  • ELISA 1:20000 - 1:100000
  • Fluorophore-linked immunosorbent assay
  • Immunocytochemistry/ Immunofluorescence
  • Immunohistochemistry 1:1000 - 1:5000
  • Western Blot 1:2000 - 1:10000
• Application Notes: This product is tested by western blot and suitable for use in immunoelectrophoresis, western-blot, competitive western-blot, ELISA and competitive ELISA assays. Specific conditions for reactivity and signal detection should be optimized by the end user.

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 (NBP1-72741)(1)

Publication using NBP1-72741

• LaRock DL, Johnson AF, Wilde S Et al. Group A Streptococcus induces GSDMA-dependent pyroptosis in keratinocytes Nature 2022-05-11 [PMID: 35545676] (WB)