DNA encoding PF3D7_1372300 (residues 27-206) and PF3D7_0800200 (residues 2,466-2,858) were PCR-amplified from P. falciparum cDNA using pairwise primers listed in Supplementary Table S1 . The PF3D7_1372300 and PF3D7_0800200 PCR products were cloned into the pET-28a vector, while the PF3D7_1372300 PCR product was cloned into the pGEX-4T-1 vector. Constructs were expressed in Escherichia coli BL21 (DE3) cells as His-tagged and GST-tagged recombinant proteins and were purified according to the manufacturer’s instructions (Ramaprasad et al., 2012 (link)). SDS-PAGE and Western blotting were used to evaluate the purified recombinant proteins. Two female New Zealand white rabbits and four female Sprague Dawley rats were immunized with His-tagged recombinant proteins emulsified with Freund’s adjuvant. Immunizations were performed four times every 2 weeks, and antisera were collected 10 days after the final immunization. IgGs were purified from the immune sera using Protein G Sepharose 4 Fast Flow Resin (GE Healthcare) and Protein A Sepharose 4 Fast Flow resin (GE Healthcare), respectively. The total proteins of iRBCs and RBCs were used to detect the specificity of the antibodies by Western blotting.
Protein a sepharose 4 fast flow resin
Manufactured by GE Healthcare
Sourced in Sweden
Protein A Sepharose 4 Fast Flow resin is a chromatography medium used for the purification of monoclonal antibodies and other Fc-containing proteins. The resin consists of recombinant Protein A coupled to Sepharose 4 Fast Flow, a high-performance agarose matrix. The Protein A ligand selectively binds to the Fc region of immunoglobulins, allowing for effective capture and separation of target proteins.
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Lab products found in correlation
2 protocols using protein a sepharose 4 fast flow resin
1
Recombinant Protein Expression and Antibody Production
2
Purification of Recombinant GA733-2-Fc Proteins
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Recombinant GA733-2-Fc and GA733-2-Fc-KDEL were purified by affinity chromatography using Protein A Sepharose 4 Fast Flow resin (GE Healthcare, Uppsala, Sweden) according to the manufacturer's recommendations. Protein extracts were dialyzed with binding buffer (20 mM sodium phosphate buffer, pH 7.0) and applied to a column containing Protein A Sepharose 4 Fast Flow resin. Weakly bound contaminating proteins were washed from the beads using binding buffer. Recombinant GA733-2-Fc and GA733-2-Fc-KDEL were then eluted with 0.1 M glycine (pH 2.9) and dialyzed in phosphate-buffered saline (PBS, pH 7.4). Protein concentrations were determined using a Bradford Protein Assay Kit (Bio-Rad, Hercules, CA, USA) with bovine serum albumin as a standard.
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