Hitrap protein a 5 ml column

Manufactured by GE Healthcare

The Hitrap Protein A 5 mL column is a lab equipment product designed for protein purification. It is a pre-packed, ready-to-use affinity chromatography column that utilizes Protein A ligand to capture and purify immunoglobulins and other Fc-containing proteins from complex samples.

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Lab products found in correlation

Superdex 200 column, by GE Healthcare (4 mentions) Histrap hp 5 ml column, by GE Healthcare (2 mentions) Pcaggs expression vector, by Addgene (2 mentions) Superdex 75 column, by GE Healthcare (2 mentions) Histrap hp, by GE Healthcare (1 mentions) Histrap excel 5 ml column, by GE Healthcare (1 mentions) Hitrap igm purification hp, by GE Healthcare (1 mentions) Superose 6 increase 10 300 gl, by GE Healthcare (1 mentions)

Close spelling variants of this product

5-ml protein A column HiTrap Protein A Protein A column HiTrap columns

4 protocols using hitrap protein a 5 ml column

Recombinant Protein Purification and Validation

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The recombinant proteins contained in the pCAGGS vector were expressed in HEK293T cells. After transfection for about three days, the supernatants of the cells were collected. The antigen and mAb proteins were initially isolated by the Histrap Hp or Hitrap Protein A 5 mL column (GE Healthcare). Then, the samples were further purified by molecular size using the Superdex 200 column (GE Healthcare) in a buffer composed of 20 mM Tris-HCl (pH 8.0) and 150 mM NaCl. The purity of the protein was determined by SDS-polyacrylamide gel electrophoresis (SDS-PAGE).
All of the antibodies have been validated in previous studies both by binding to SARS-CoV-2 spike or influenza HA1, and when applicable, have been confirmed to give similar results as that described in publications by other groups.

Shi R., Zeng J., Xu L., Wang F., Duan X., Wang Y., Wu Z., Yu D., Huang Q., Yao Y.G, & Yan J. (2022). A combination vaccine against SARS-CoV-2 and H1N1 influenza based on receptor binding domain trimerized by six-helix bundle fusion core. eBioMedicine, 85, 104297.

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Monkeypox Virus Antigen Purification

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The optimized sequences of mpox virus H3, M1, A29, A35, B6, E8, and VACV H3, L1, A27, D8, B5, A33 fused with N-terminal native signal peptides and C-terminal 8× His tag respectively, then cloned into the pCAGGS expression vector (Addgene). The plasmids were transiently transfected into HEK293T cells. After 5 days, the supernatant was collected and purified by a HisTrap HP 5 ml column (GE Healthcare). Each sample was purified via size-exclusion chromatography with a Superdex 75 column (GE Healthcare) in PBS. To prepare antibodies against the antigens of mpox, the variable region links to the sequence encoding the constant region of mouse IgG2a, resulting in a full-length H chain and L chain plasmid. MAbs expressed by co-transfection of HEK293T cells. The antibodies were isolated by Hitrap Protein A 5 mL column (GE Healthcare) and purified by Superdex 200 column (GE Healthcare) in PBS.

Zeng J., Li Y., Jiang L., Luo L., Wang Y., Wang H., Han X., Zhao J., Gu G., Fang M., Huang Q, & Yan J. (2023). Mpox multi-antigen mRNA vaccine candidates by a simplified manufacturing strategy afford efficient protection against lethal orthopoxvirus challenge. Emerging Microbes & Infections, 12(1), 2204151.

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Engineered Nanobody-Fc Antibody Constructs

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The dimer constructs (DR14 and DS43) were engineered via fusing nanobody to hFc. The homo-trimer versions of R14 and S43 (TR14 and TS43) were constructed via head-to-tail with (GGGGS)₃ linkers and a C-terminal His-tag. The coding sequence of R14 or S43 fused to the Fc of human IgM was cloned into the pCAGGS vector to generate IgM versions of the antibodies (MR14 and MS43). The coding sequence of TR14 was synthesized by Nanjing GenScript Biotech Co., Ltd., and TS43 was synthesized by Tsingke Biotechnology Co., Ltd. DR14 and DS43 were expressed in Freestyle 293F cells and were purified using a HiTrap Protein A 5-mL column (GE Healthcare) and Superdex 200 column (GE Healthcare). TR14 and TS43 were expressed in Freestyle 293F cells and were purified using a HisTrap EXCEL 5-mL column (GE Healthcare) and Superdex 200 column (GE Healthcare). The recombinant plasmid for MR14 or MS43 and a human J-chain expressing vector were co-transfected into Freestyle 293F cells to express the MR14 or MS43 proteins, which were further purified by HiTrap™ IgM Purification HP (GE Healthcare) and Superose 6 Increase 10/300 GL (GE Healthcare) chromatography.

Liu H., Wu L., Liu B., Xu K., Lei W., Deng J., Rong X., Du P., Wang L., Wang D., Zhang X., Su C., Bi Y., Chen H., Liu W.J., Qi J., Cui Q., Qi S., Fan R., Jiang J., Wu G., Gao G.F, & Wang Q. (2023). Two pan-SARS-CoV-2 nanobodies and their multivalent derivatives effectively prevent Omicron infections in mice. Cell Reports Medicine, 4(2), 100918.

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Monkeypox Virus Antigen Purification

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