For research use only!
Shipping: shipped at ambient temperature
Storage Conditions: store at 4 °C
Shelf Life: 12 months after date of delivery
Form: 4 % crosslinked agarose supplied as a suspension containing 20 % ethanol
Protein A resin allows batch or column purification of classes, subclasses and fragments of immunoglobulins from cell culture media and biological fluids. Recombinant Protein A is covalently bound to the matrix, avoidin protein loss and allowing re-use of the resin.
Degree of substitution: 3 mg rProtein A / ml resin
Binding capacity: 25 mg human IgG / ml resin
Protein A consists of a single polypeptide chain which contains five highly homologous antibody-binding domains. The binding site is located on the Fc region of immunoglobulin. Protein A has affinity for IgG from a variety of mammalian species and for some IgA and IgM. Recombinant Protein A shares identical binding properties to IgG as the Cowan I strain of natural Protein A.
1. Removal of the storage solution
Wash the beads with 5 - 10 column volumes of distilled water to remove the storage solution.
Please note: For batch purification, remove the storage solution by washing the product on a medium porosity sintered glass funnel.
2. Equilibration of the Protein A Agarose resin
Equilibrate the column with 5 - 10 column volumes of binding buffer.
Binding buffer: IgG from most species binds at neutral pH. The buffers used most frequently are sodium phosphate (25 mM) or Tris (50 mM) at pH 7.0. Binding occurs through an induced hydrophobic frit and is promoted by addition of salts. At alkaline pH, the interaction between Protein A and the antibody is stronger. Other buffers used frequently are PBS (100 mM), NaCl (150 mM) at pH 7.2.
3. Application of the sample
Once the resin is equilibrated, the sample containing the immunoglobulin for purification is applied.
4. Washing of the Protein A Agarose resin
Wash with the binding buffer until OD280 reaches baseline level.
5. Elution of the pure immunoglobulin
Elution is usually achieved at reduced pH. Depending on the sample it may be necessary to decrease pH below 3.0. Most immunoglobulins are eluted in glycine (100 mM) or citric acid buffer (100 mM) at pH 3.0.
Please note: It is recommended to add 0.15 ml of a buffer at pH 9.0 (e.g 1 M Tris) per ml of purified immunoglobulin to neutralize the eluted fractions. A more drastic method uses potassium isothiocyanate (3 M) as elution buffer.
The following procedure describes how a column is packed from bulk affinity resins:
Maximum flow rate: 500 cm/h
Maximum pressure: 21.8 psi (1.5 bar)