Antibody Purification

A wide variety of methods are used to purify antibodies. The correct choice of purification method depends upon the class/subclass of the antibody, the species in which it was raised and the intended use of the antibodies. Antibodies from tissue culture supernatant, serum or ascites can be purified using one or more of the following methods offered by Covalab:

Immunoaffinity purifaction Protein A/G purification Ion exchange chromatography Procedures and methodology used for the purification and a Quality Control report (including analysis of yield, purity and immunoreactivity) are provided with each purified product

Type of sample:
- Serum
- Ascitic fluid
- Cell culture supernatant

Isotype: IgG

Average concentration:
- Serum: 1 to 3 mg/mL
- Ascitic fluid: 1 to 5 mg/mL
- Cell culture supernatant: 5 to 20 µg/mL

This service includes purification by chromatography, control of purified product by PAGE, equilibration in PBS buffer containing 0.02% sodium azide.

Steps:
• Loading of the antiserum on a Protein A or G column and elution of bound antibodies
• SDS-PAGE analysis of purified antibodies

• IgY purification from egg yolk

Chicken antibodies IgY are the equivalent to mammalian IgG antibodies and can be isolated from serum or egg yolk. The egg yolks contain at least the same concentration of IgY antibodies than found in chicken or rabbit serum. IgY can be used in most biochemical and cellular applications, including western blots. As the Fc region of chicken IgY is sufficiently different from mammalian IgG chicken antibodies do not cross-react with mammalian proteins, do not bind with mammalian rheumatoid factors or Fc-receptors and with proteins A or G. Therefore the background signal or false response in certain immunochemical assays is reduced.

• Extraction of a protein fraction containing IgY

• Instead of sending whole eggs, we propose to extract a protein fraction containing IgY from egg yolks and send this entire fraction to the client who can then purify the IgY directly from the precipitate.

• Purification of IgY

This service consists in purifying IgY from yolk eggs by selective precipitation followed by ion exchange chromatography (Carroll SB et Stollar BD. Antibodies to Calf thymus RNA polymerase II from egg yolks of immunized hens. (1983) J Biol Chem. 1983 jan 10; 253(1):24-6). At the end of the purification the purity of antibodies is checked by PAGE.

• Note: It has generally been found to be about 80%.
  
  Steps:
  • Preparation of the egg yolk extract by two-step precipitation
  • Loading of the egg yolk extract on the ion exchange column and elution of bound antibodies
  • ELISA testing on starting material and immunopurified antibodies
  • SDS-PAGE analysis of purified antibodies
  
  Yield of purification: ~ 10 to 15 mg of IgY / egg yolk

Any questions? Please contact us