Antigens gp43 [29 (link)] and cell-free antigen (CFA) [30 (link)] were obtained as previously described. Briefly, gp43 was purified at the Laboratory of Mycology from the Institute of Biomedical Sciences (University of Sao Paulo, Sao Paulo, SP, BR) and at the Laboratory of Medical Investigation in Immunology (HCFMUSP) from the exoantigen of P. brasiliensis yeast B-339 strain through an adsorbent column with murine monoclonal antibody anti-gp43 coupled to an Affi-Gel 10 column (Bio-Rad, Hercules, CA, USA). Gp43 was eluted with acid buffer (pH 2.8) and neutralized with 1M Tris (pH 9.0). The material was concentrated in a 10K Amicon apparatus (Merck Millipore, Darmstadt, Germany). CFA was obtained by mixing a suspension of P. brasiliensis yeast B-339 strain in RPMI 1640 medium (Gibco, Grand Island, NY, USA) in a vortex mixer and immediately centrifuging at 10,000× g. The resulting supernatant fluid contained the antigen. Both gp43 and CFA had their protein contents determined by the Bradford method [31 (link)], confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) [32 (link)], and stored at −80 °C until use.
Affi gel 10 column
Manufactured by Bio-Rad
Sourced in United States
Affi-Gel 10 column is a pre-packed chromatography column designed for affinity-based purification of proteins. It features a hydrophilic agarose gel matrix that allows for gentle and efficient separation of target biomolecules. The column is suitable for a variety of affinity-based techniques, including immunoaffinity, metal-chelate, and ligand-binding purifications.
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Lab products found in correlation
2 protocols using affi gel 10 column
1
Purification and Characterization of Paracoccidioides Antigens
2
Generating EphA2 S901 Phospho-Specific Antibodies
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The peptide KLPSTSGpSEGVPFR, corresponding to EphA2 residues 895–907 with S901 phosphorylated and an added N-terminal lysine, was coupled to BSA using glutaraldehyde and used to immunize rabbits. Antibodies were then affinity purified from immune serum using peptide coupled to an Affi-Gel 10 column (1536046; Bio-Rad Laboratories, Hercules, CA). Antibodies purified using the phosphorylated or the nonphosphorylated peptide preferentially recognize EphA2 phosphorylated on S901. They recognize EphA2 WT overexpressed by transient transfection in HEK293 cells but not EphA2 that has been treated with calf intestinal alkaline phosphatase, which indiscriminately dephosphorylates pS/pT/pY residues (Figure 3F ). The antibodies also do not recognize the EphA2 S901A mutant, which lacks the S901 phosphorylation site (Figure 3G ). These data confirm the desired specificity of the antibodies for the phosphorylated S901 motif.
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