Donkey F(ab')2 anti-Mouse IgG (H+L) Secondary Antibody [PE] (Pre-adsorbed)

Dot Blot: Donkey F(ab')2 anti-Mouse IgG (H+L) Secondary Antibody [PE] (Pre-adsorbed) [NB7596] - Dot Blot of Donkey F(ab')2 anti-Mouse IgG (H+L) Secondary antibody [PE] (Pre-adsorbed). Antigen: Mouse IgG. Load: 100 ng, 33.3 ng, 11.1 ng, 3.7 ng or 1.23 ng as indicated.Primary antibody: N/A.Secondary antibody: Donkey F(ab')2 anti-Mouse IgG (H+L) Secondary antibody [PE] (Pre-adsorbed) at 1:1,000 for 60 min at RT.Block: Blocking Buffer for Fluorescent Western Blotting for 60 min at RT.

Dot Blot: Donkey F(ab')2 anti-Mouse IgG (H+L) Secondary Antibody [PE] (Pre-adsorbed) [NB7596] - Dot Blot results of Donkey F(ab')2 anti-Mouse IgG (H+L) Secondary antibody [PE] (Pre-adsorbed).Dots are Mouse IgG at (1) 100ng, (2) 33.3ng, (3) 11.1ng, (4) 3.70ng, (5) 1.23ng. Blocking buffer for 60 min at RT.Primary Antibody: Donkey F(ab')2 Anti-Mouse IgG Antibody Phycoerythrin at 1ug/mL for 1hr at RT.Secondary Antibody: none.Imaged with ChemiDoc, Rhodamine filter.

Dot Blot of F(ab')2 Donkey Anti-Mouse IgG Antibody Phycoerythrin conjugated Min X Bv Ch Gt GP Ham Hs Hu Rb Rt & Sh Serum Proteins. Antigen: Mouse IgG. Load: 100 ng, 33.3 ng, 11.1 ng, 3.7 ng or 1.23 ng as indicated. Primary antibody: N/A. Secondary antibody: F(ab')2 Donkey Anti-Mouse IgG Antibody Phycoerythrin conjugated Min X Bv Ch Gt GP Ham Hs Hu Rb Rt & Sh Serum Proteins at 1:1,000 for 60 min at RT. Block: Blocking Buffer for Fluorescent Western Blotting () for 60 min at RT.

• Reactivity: Mu
• Applications: WB, DB, Flow, ICC/IF, IHC
• Clonality: Polyclonal
• Host: Donkey
• Conjugate: PE
• Format: Pre-adsorbed
• Concentration: LYOPH

Donkey F(ab')2 anti-Mouse IgG (H+L) Secondary Antibody [PE] (Pre-adsorbed) Summary

• Description: Store vial at 4C prior to restoration. Restore with deionized water (or equivalent). This product is stable at 4C as an undiluted liquid. Dilute only prior to immediate use. Centrifuge product if not completely clear after standing at room temperature. Do not freeze after reconstitution. Store reagent in the dark. Use subdued lighting during handling and incubation of cells prior to analysis.
  
  This product was prepared from monospecific antiserum by immunoaffinity chromatography using Mouse IgG coupled to agarose beads followed by solid phase adsorption(s) to remove any unwanted reactivities, pepsin digestion and chromatographic separation. Assay by immunoelectrophoresis resulted in a single precipitin arc against anti-Phycoerythrin, anti-Donkey Serum, Mouse IgG and Mouse Serum
• Immunogen: Donkey F(ab')2 anti-Mouse IgG (H+L) Secondary Antibody [PE] (Pre-adsorbed) was produced by repeated immunization with Mouse IgG whole molecule in donkey.
• Specificity: No reaction was observed against anti-Pepsin, anti-Donkey IgG F(c), Bovine, Chicken, Goat, Guinea Pig, Hamster, Horse, Human, Rabbit, Rat or Sheep Serum Proteins.
• Isotype: IgG
• Clonality: Polyclonal
• Host: Donkey
• Purity: Multi-step

Applications/Dilutions

• Dilutions:
  • Dot Blot
  • Flow Cytometry 1:100 - 1:250
  • Immunocytochemistry/ Immunofluorescence 1:100 - 1:250
  • Immunohistochemistry 1:10-1:500
  • Immunohistochemistry-Paraffin 1:200-1:500
  • Western Blot 1:100-1:500
• Application Notes: This product has been tested by dot blot and is suitable for immunomicroscopy and flow cytometry or FACS analysis as well as other antibody based fluorescent assays requiring extremely low background levels, absence of F(c) mediated binding, lot-to-lot consistency, high titer and specificity. The maximum amount of reagent required to stain 1 x 10E6 cells in flow cytometry is approximately 1.0 ug of antibody conjugate. Lesser amounts of reagent may be sufficient for staining. Optimal titers for other applications should be determined by the researcher. As a general guideline dilutions of 1:100 to 1:250 should be suitable for most applications.

Packaging, Storage & Formulations

• Storage: Store lyophilized antibody at 4C. Aliquot reconstituted liquid and store at 4C in the dark. Do not freeze.
• Buffer: Lyophilized from 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
• Concentration: LYOPH
• Purity: Multi-step
• Reconstitution Instructions: Reconstitute with 1.0 ml deionized water (or equivalent).

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.