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Protein a agarose resin

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Protein A agarose resin is a solid-phase affinity chromatography medium used for the purification of antibodies and Fc-containing proteins. It consists of Protein A, a bacterial cell wall protein, covalently coupled to agarose beads. Protein A has a high affinity for the Fc region of immunoglobulins, allowing for the selective capture and purification of antibodies from complex mixtures.

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Lab products found in correlation

Protease lys c, by Merck Group (2 mentions) Polypropylene column, by Thermo Fisher Scientific (1 mentions) Amicon ultra 4 spin columns, by Merck Group (1 mentions)

Spelling variants of this product

Protein A resin (30 citations) Protein A agarose (6 citations)

5 protocols using protein a agarose resin

1

Detailed Fab Fragment Purification

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The 5B3 Fab was obtained by fragmentation of mouse 5B3 IgG using Pierce Mouse IgG1 Fab and F(ab’)2 preparation kits according to the manufacturer’s protocol.
The h5B3.1 Fab fragment was obtained by fragmentation of h5B3.1 IgG with Lys-C protease (EMD Millipore) and affinity purification using protein A agarose resin (Genscript). Briefly, 1.0 mg IgG was incubated with 0.5 μg Lys-C for 7 hours at 37°C. The reaction was quenched by addition of PMSF to 1 mM final concentration and the sample was purified using a protein A resin.
The Fab-containing flow-through was concentrated and further purified using a Superdex 75 10/300 gel filtration column equilibrated in a buffer containing 50 mM Tris pH 8.0 and 150 mM NaCl.
Dang H.V., Chan Y.P., Park Y.J., Snijder J., Da Silva S.C., Vu B., Yan L., Feng Y.R., Rockx B., Geisbert T.W., Mire C.E., Broder C.C, & Veesler D. (2019). An antibody against the F glycoprotein inhibits Nipah and Hendra virus infections. Nature Structural & Molecular Biology, 26(10), 980-987.
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2

Expi293 Antibody Production Protocol

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The Expi293 expression system was used for antibody production according to the manufacturer’s instructions. A 2:1 ratio of light chain to heavy chain plasmid was used in transfections. mAb-containing supernatants from transfected expi293 cells were clarified by centrifugation and then incubated with protein A agarose resin (Genscript) in batch format overnight, followed by washing, elution, and buffer exchange into DPBS as described (Smith et al., 2009 (link)). Antibodies used in vivo were verified endotoxin-free using a commercial kit (ThermoFisher). Note that all antibodies produced in this study were expressed as human IgG1.
Davis C.W., Jackson K.J., McElroy A.K., Halfmann P., Huang J., Chennareddy C., Piper A.E., Leung Y., Albariño C.G., Crozier I., Ellebedy A.H., Sidney J., Sette A., Yu T., Nielsen S.C., Goff A.J., Spiropoulou C.F., Saphire E.O., Cavet G., Kawaoka Y., Mehta A.K., Glass P.J., Boyd S.D, & Ahmed R. (2019). Longitudinal Analysis of the Human B Cell Response to Ebola Virus Infection. Cell, 177(6), 1566-1582.e17.
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3

Expi293 System for Antibody Production

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The Expi293 expression system was used for antibody production according to the manufacturer’s instructions. A 2:1 ratio of light chain to heavy chain plasmid was used in transfections. mAb-containing supernatants from transfected expi293 cells were clarified by centrifugation and then incubated with protein A agarose resin (Genscript) in batch format overnight, followed by washing, elution, and buffer exchange into DPBS as described (Smith et al., 2009 (link)). Antibodies used in vivo were verified endotoxin-free using a commercial kit (ThermoFisher). Note that all antibodies produced in this study were expressed as human IgG1.
Davis C.W., Jackson K.J., McElroy A.K., Halfmann P., Huang J., Chennareddy C., Piper A.E., Leung Y., Albariño C.G., Crozier I., Ellebedy A.H., Sidney J., Sette A., Yu T., Nielsen S.C., Goff A.J., Spiropoulou C.F., Saphire E.O., Cavet G., Kawaoka Y., Mehta A.K., Glass P.J., Boyd S.D, & Ahmed R. (2019). Longitudinal Analysis of the Human B Cell Response to Ebola Virus Infection. Cell, 177(6), 1566-1582.e17.
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4

Monoclonal Antibody Fab Fragment Purification

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The 5B3 Fab was obtained by fragmentation of mouse 5B3 IgG using Pierce mouse IgG1 Fab and F(ab′)2 preparation kits according to the manufacturer’s protocol.
The h5B3.1 Fab fragment was obtained by fragmentation of h5B3.1 IgG with Lys-C protease (EMD Millipore) and affinity purification using protein A agarose resin (Genscript). Briefly, 1.0 mg IgG was incubated with 0.5 µg Lys-C for 7 h at 37 °C. The reaction was quenched by addition of PMSF to 1 mM final concentration and the undigested and Fc-containing portion of the sample was removed using a protein A resin. The Fab-containing flow-through from the protein A affinity step was collected.
The Fab-containing fraction was concentrated and further purified using a Superdex 75 10/300 gel filtration column equilibrated in a buffer containing 50 mM Tris pH 8.0 and 150 mM NaCl.
Dang H.V., Chan Y.P., Park Y.J., Snijder J., Da Silva S.C., Vu B., Yan L., Feng Y.R., Rockx B., Geisbert T.W., Mire C.E., Broder C.C, & Veesler D. (2019). An antibody against the F glycoprotein inhibits Nipah and Hendra virus infections. Nature Structural & Molecular Biology, 26(10), 980-987.
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5

Purification of Recombinant AG8 Antibody

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The pMAZ-AG8H and pMAZ-AG8L plasmids, which encode the heavy and light chains of AG8 IgG, respectively, were constructed using an eCube Plasmid DNA Mini Kit (PhileKorea, Seoul, Korea) and transfected into Expi293 cells using polyethyleneimine, as described in the literature26 . After resuspension of the cells in 300 ml of GIBCO FreeStyleTM medium (Thermo Fisher Scientific, Waltham, MA, USA), incubation at 37 °C with shaking at 125 rpm under 8% CO2 for 6 days, and centrifugation at 4000×g, the supernatants were mixed with 40 ml of 25 × PBS (pH 7.4) and 1 ml of a slurry of Protein A agarose resin (GenScript, Piscataway, NJ, USA). The resuspension was incubated at 4 °C for 16 h and passed through a polypropylene column (Thermo Fisher Scientific, Waltham, MA, USA) to recover the resin. Next, 100 ml of 1× PBS (pH 7.4) was added to the column to wash the resin, and 3 ml of 100 mM glycine-HCl buffer (pH 2.5) was loaded onto the column for elution. The eluents were immediately neutralized by the addition of 1 ml of Tris-Cl (pH 8.0). After buffer exchange with 1× PBS (pH 7.4) using Amicon Ultra®4 spin columns (Merck Millipore; 3-kDa cutoff), the concentration and purity of AG8 IgG were analyzed by measuring the absorbance at 280 nm and by 4–15% SDS–PAGE.
Ju M.S., Ahn H.M., Han S.G., Ko S., Na J.H., Jo M., Lim C.S., Ko B.J., Yu Y.G., Lee W.K., Kim Y.J, & Jung S.T. (2021). A human antibody against human endothelin receptor type A that exhibits antitumor potency. Experimental & Molecular Medicine, 53(9), 1437-1448.
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