Rprotein a sepharose fast flow beads

Manufactured by Cytiva

RProtein A Sepharose® Fast Flow beads are high-performance agarose-based chromatography media designed for the purification of antibodies and other proteins that bind to Protein A. The beads feature a rigid, highly cross-linked agarose matrix that provides excellent mechanical strength and flow properties, enabling efficient and high-throughput purification.

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Lab products found in correlation

Expifectamine 293 transfection kit, by Thermo Fisher Scientific (3 mentions) Poly prep chromatography column, by Bio-Rad (2 mentions) Amicon ultra 4 centrifugal filter, by Merck Group (2 mentions) Dulbecco phosphate buffered saline (dpbs), by Thermo Fisher Scientific (1 mentions) Superdex 200 increase 5 150 gl column, by Cytiva (1 mentions)

3 protocols using rprotein a sepharose fast flow beads

Generation of Anti-SARS-CoV-2 RBD Antibodies

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The top-ranked enriched IgG clones were selected and cDNAs of relative variable region of paired heavy- and light-chain were codon-optimized and cloned separately into human IgG1 heavy chain and light chain expression vectors, containing the human IgG1 constant regions (pFuse plasmids). IgG1 antibodies were expressed in Expi293F^™ cells. ExpiFectamine 293 transfection kit (Thermo fisher) was utilized for heavy and light chain plasmids transfection following the manufacturer’s instruction. After 5 days, the antibody containing supernatants were collected. Suitable amount of rProtein A Sepharose® Fast Flow beads (Cytiva) was pre-washed and added into supernatants. After overnight incubation at 4 °C, antibody bound protein A beads were collected with Poly-Prep® Chromatography Columns (BIO-RAD). After 3 times wash with DPBS, mAbs were eluted with Fab elution buffer, then neutralized with Tris-HCl. Buffer exchange was performed with Amicon Ultra-4 Centrifugal Filter (MilliporeSigma) to keep mAbs in PBS for following assays. The numbering of mAbs was based on the order of mouse immunization and cloning. Clones 1–4 were mAbs chosen from enriched clones from RBD-his tag protein immunized C57BL/6J mice. Clone 5–11 were mAbs chosen from RBD-his tag protein immunized BALB/c mice.

Peng L., Hu Y., Mankowski M.C., Ren P., Chen R.E., Wei J., Zhao M., Li T., Tripler T., Ye L., Chow R.D., Fang Z., Wu C., Dong M.B., Cook M., Wang G., Clark P., Nelson B., Klein D., Sutton R., Diamond M.S., Wilen C.B., Xiong Y, & Chen S. (2021). Monospecific and bispecific monoclonal SARS-CoV-2 neutralizing antibodies that maintain potency against B.1.617. bioRxiv.

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Production and Purification of Monoclonal Antibodies

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The top-ranked enriched IgG clones were selected and cDNAs of a relative variable region of paired heavy- and light-chain were codon-optimized and cloned separately into human IgG1 heavy chain and light chain expression vectors, containing the human IgG1 constant regions (pFuse plasmids). IgG1 antibodies were expressed in Expi293F^TM cells. ExpiFectamine 293 transfection kit (Thermo Fisher) was utilized for heavy and light chain plasmids transfection following the manufacturer’s instruction. After 5 days, the antibody-containing supernatants were collected. A suitable amount of rProtein A Sepharose^® Fast Flow beads (Cytiva) was prewashed and added into supernatants. After overnight incubation at 4 °C, antibody-bound protein A beads were collected with Poly-Prep^® Chromatography Columns (BIO-RAD). After three times wash with DPBS, mAbs were eluted with Fab elution buffer, then neutralized with Tris-HCl. Buffer exchange was performed with Amicon Ultra-4 Centrifugal Filter (MilliporeSigma) to keep mAbs in PBS for the following assays. The numbering of mAbs was based on the order of mouse immunization and cloning. Clones 1–4 were mAbs chosen from enriched clones from RBD-his tag protein immunized C57BL/6 J mice. Clone 5-11 were mAbs chosen from RBD-his tag protein immunized BALB/c mice.

Peng L., Hu Y., Mankowski M.C., Ren P., Chen R.E., Wei J., Zhao M., Li T., Tripler T., Ye L., Chow R.D., Fang Z., Wu C., Dong M.B., Cook M., Wang G., Clark P., Nelson B., Klein D., Sutton R., Diamond M.S., Wilen C.B., Xiong Y, & Chen S. (2022). Monospecific and bispecific monoclonal SARS-CoV-2 neutralizing antibodies that maintain potency against B.1.617. Nature Communications, 13, 1638.

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Recombinant mAb Production and Purification

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The cDNAs of paired heavy- and light-chains from top-enriched IgG clonotypes were codon-optimized and respectively subcloned into human IgG1 expression vectors, based on Gibson assembly, to generate recombinant mAbs. mAbs were produced by transient transfection into Expi293F^™ cells with equal amounts of paired heavy- and light-chain expression vectors using the ExpiFectamine 293 transfection kit according to the manufacturer’s protocol (Thermo Fisher). Five days post antibody expression, the secreted mAbs from cultured cells were collected and purified by affinity chromatography on rProtein A Sepharose Fast Flow beads according to the manufacturer’s instructions (Cytiva). Eluted mAbs were further purified by size exclusion chromatography (SEC) using a Superdex 200 Increase 5/150 GL column (Cytiva), and the column was pre-equilibrated in 1x DPBS (Thermo). The purified mAbs were examined by running SDS-PAGE and kept in −80°C until needed.

Ren P., Peng L., Yang L., Suzuki K., Fang Z., Renauer P.A., Lin Q., Bai M., Li T., Clark P., Klein D, & Chen S. (2023). RAMIHM generates fully human monoclonal antibodies by rapid mRNA immunization of humanized mice and BCR-seq. Cell chemical biology, 30(1), 85-96.e6.

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