Protein A-sepharose

Suspend the matrix in 20 mL PBS and collect the protein A-Sepharose by centrifugation at 500g for 5 min. Aspirate the supernatant. Repeat this wash step three times with 20 mL PBS. 

Bywater, R., Eriksson, G.-B., and Ottosson, T. (1983) Desorption of immunoglobulins from Protein A-Sepharose C1-4B under mild conditions. J. Immunol. Methods 64, 1-6. 

Ey, P. L., Prowse, S. J., and Jemkin, C. R. (1978) Isolation of pure IgGl, lgG2a and lgG2b immunoglobulins from mouse serum using Protein A-Sepharose. Immunochemistry 15, 429 36. 

Ey, P.L., Prowse, S.J., and Jenkin, C.R., Isolation of pure IgG, IgGj and IgG2(, immunoglobulins from mouse serum using protein A-Sepharose, Immunochemistry, 15, 429436, 1978. 

Protein A-Sepharose can be used to purify antibody fragments encoded by VH segments from the VH3 family (17). MAb 9E10 see Section 3.7.) can be coupled to CNBr-activated Sepharose according to the manufacturer s instructions and used to purify myc-tagged fragments see Note 11). 

Pre-swell the protein A-Sepharose (or protein A-9E10-Sepharose) in PBS. 

Fill a 10-mL plastic column with about 1 mL protein A-Sepharose, and equilibrate column with 50 mL PBS. 

For small-scale purification, mix 5—10 mL of antibody fragments with 100-200 pL of pre-swollen protein A-Sepharose. Purify using ultrafree-MC filters following the above protocol, but wash with 1 mL of buffer, and elute with 100 pL of eluant... 

In addition, 9E10 immobilized on protein A-Sepharose using dimethyl pimelim-idate can be used for purification of these antibody fragments (15)... 

Saunders et al. (1997) have raised a polyclonal antiserum using a peptide specific for ER 3. The peptide (CLSKAKRNGGHAPRVLEL) corresponding to amino acids 196-213 of rat ER(3 was conjugated to keyhole limpet hemocyanin and used to immunize rabbits according to standard procedures. Polyclonal IgGs were purified from serum on a Hitrap protein A Sepharose column based on the manufacturer s instruction (Pharmacia). 

Fig. 2. Analysis of human B-receptors purified by single step immunoaflinity chromatography from T47D cytosols. B-receptors were purified by circulating cytosols from T47D cells over a column prepared by cross-linking PR-6 to protein-A-Sepharose. Proteins bound to the column were eluted with buffer at pH 11.5. Eluates were separated by SDS-PAGE. and the gels were silver stained or blotted to nitrocellulose and analyzed with PR-6.

May detect other lipid kinases such as PI 4-kinases that coassociate with immunoprecipitated proteins or nonspecifically associate with protein A-Sepharose beads. 

Add antibody-Protein A Sepharose complex to pre-cleared cell lysate. Incubate for 2 h on ice, on rotation. 

Purification of antisera (10 ml) for mass spectrometric epitope analysis was per-formedon Protein A-Sepharose (Amersham Biosciences) according to manufacturer s instructions. Protein quantification was performed using the micro-BCA kit (Pierce, Rockford) positive elution fractions were concentrated to 7 (ig/ml. 

Fig. 6. After chromatography on DEAE cellulose, a preparation of Fab from goat IgG (well 2) still had material reacting with anti-Fc (well 1). Following passage through a column of protein A-Sepharose, the purified Fab (well 3) no longer had material reacting with the anti-Fc (well 1) but still reacted with anti-Fab (well 4).

Five grams of protein A-Sepharose (Pharmacia Fine Chemicals, AB, Uppsala, Sweden) are rehydrated according to the manufacturer s directions, packed into a column, and washed with 10 mM phosphate-buffered 0.15 M NaCl, pH 7.4. Goat Fab (23 mg in 13 ml) containing some molecules with Fc determinants still present (Fig. 6) was passed through the column. The effluent contained Fab but no longer reacted with anti-Fc (Fig. 6). 

Purification of Immunoglobulins and Anti-immunoglobulins. The IgG fraction of an antiserum can be purified according to any of a number of established techniques. If the antiserum was raised in rabbits, the most convenient purification procedure to use is affinity chromatography on protein A-Sepharose (Pharmacia Fine Chemicals, Uppsala, Sweden). Excellent yield and purity is obtained by following the recommendations of the manufacturer. 

Purification of Antibodies Using Protein A-Sepharose and FPLC... 

Protein A-Sepharose Chromatography by Fast Protein Liquid Chromatography (FPLC)... 

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