Protein A/G Purification Column
First Edition (Revised on April, 2016)
Staphylococcal protein A is a kind of cell wall protein from Staphylococcus aureus , it can be combined with the Fc region of mammalian IgG. Recombinant protein A and CNBr activated beaded-form of agarose were coupled to made prepacked columns of protein A , it can specifically combined with Fc section of a variety of immunoglobulin.
Prepacked columns of protein A can be used for the purification of antibodies from different species and subtypes, and it could also be used for capturing immune complexes in Co-Immunoprecipitation. The product uses the multilocus crosslinking technology, it possesses good stability, high specificity and broad-spectrum binding force, 1 ml of the matrix can specifically combined with 5-10 mg of antibody.
REAGENTS AND MATERIALS PROVIDED
|Prepacked column of protein A||5 ml|
For regular use, save at 4℃. Add glycerin and placed at -20 ℃ for long-term preservation, valid for one year.
IMPORTANT PRODUCT INFORMATION
Antibody purification column can be used repeatedly, but please avoid purification of different antibodies by one column, because tight combination occur between the agar and previously antibody, and acid elution can not completely wash away the previously binding immunoglobulin, that will lead to cross-reactivity.
1. Take out the Protein A column and equilibrated to room temperature, and wash the column with PBS (10 folds of column volume).
2. After high-speed centrifugation of the serum or other body fluids which contain antibody, the supernatant is mixed with an equal volume of 2×PBS, adjusting the pH and ionic concentration. Then the liquid should be added into the column slowly.
3. Wash the column with PBS (more than 10 folds of column volume), until no protein was detected in the effluent liquid.
4. Add 0.1 M citric acid (pH 2.7) or 0.1 M glycine (pH 3.0) (2-5 folds of column volume) into the column, and collect the elution liquid.
5. Add saturated Na2HPO4 into the elution liquid for neutralization.
6. Concentrate the elution liquid to desired volume, add 50% glycerol, and save at -20 ℃;
7. The used protein A column needs to be regenerated timely, after elution of the antibody with acid, use 10 folds of column volume of PBS to neutralize the column, and then wash the column with 20% ethanol solution. For storage, seal two sides of the column, and store at 4℃.