Protein A Agarose


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Protein A Agarose

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Cat. No. : COP12
Product Overview : 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. So Protein A Agarose binds specifically to the Fc region of IgG molecules. As a result of this property, Protein A Agarose has been used to pull down antibody: antigen complexes in immunoprecipitation experiments. This Protein A Agarose is prepared by covalently coupling purified Protein A to 4% crosslinked agarose beads by a cyanogen bromide method. Immobilization of protein A agrose can be used to isolate IgG fractions from crude serum, ascites fluid or hybridoma upernatants.
Ligand ensity : approximately 6 mg Protein A per ml of drained gel.
Dynamic Binding Capacity : approximately 30-40mg rabbit IgG per ml of drained gel (calculated as the amount of rabbit IgG adsorbed to the gel before flow exceeded 1% of the absorbance of the ingoing solution at 30 cm/hour and a 0.92 mg/ml sample concentration).
Bead Structure : 4 % cross-linked agarose.
Bead Size Range : 45 to 165 μm.
Mean Bead Size : approximately 90μm.
Maximum Linear Flow Rate : >300 cm/hour.
Maximum Operating Backpressure : 0.3 MPa (3 bar).
pH Stability : 2 to 11(short term). 3 to 10(long term).
Physical stability : Negligible volume variation due to changes in pH or ionic strength.
Sanitization : Sanitize column using 70% ethanol.
Formulation : Provided as a aqueous suspension of Protein A Agarose in 20% ethanol/phosphate buffer saline containing 0.08% sodium azide.
Storage : Store refrigerated at 4-8°C. DO NOT FREEZE. Products are stable for a minimum of 1 year from date of receipt when stored at 4-8°C. Non-sterile.
Ship Conditions : Ship ambient temperature, do not freeze, refrigerate upon arrival.
General Procedure : a.Wash the beads with 5 - 10 column volumes of distilled water to remove the storage solution.b.Equilibrate the column with 5 - 10 column volumes of the wash/binding buffer. Once the column is equilibrated, the sample containing the immunoglobulin for purification is applied.c.Wash with the binding buffer until OD280 reaches baseline level. d.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. e.Once the sample has been eluted, wash the affinity matrix with 2 CV of elution buffer. Re-equilibrate the column with at least 10 CV of 1X Wash/Binding Buffer.

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