We produced recombinant hResistin protein in our laboratory using a mammalian cell expression system. C-terminal FLAG-tagged hResistin and mouse RELMα were generated by PCR, inserted into pcDNA5/FRT/TO vector, and then integrated in a Flp recombinase-dependent manner into the genome of the Flp-In™ T-Rex™ 293 cell line using the Flp-In™ T-Rex™ kit from Invitrogen as we have described (4 (link)). Production of these recombinant proteins was induced by adding tetracycline (1 μg/ml) to the cell culture media. hResistin was purified by anti-FLAG M2 antibody agarose (A2220, Sigma) column chromatography from the 293-cell culture medium with FLAG (0.1 mg/ml) elution. Presence of the protein was determined by SDS-PAGE gel Coomassie staining, and concentration of the protein in the elution was determined by the Bio-Rad Protein Assay. Activity assays were performed on each lot of protein purified. After being serum starved, human (h) PASMCs were treated with 100 nM recombinant hResistin, whereas 3T3 mouse embryonic fibroblasts were treated with the same dosage of recombinant RELMα, all for 15 min. Then cells were collected, lysed, and processed for western blotting. Activity of the purified RELM proteins was tested by assaying their ability to induce Akt phosphorylation (4 (link)).
Anti flag m2 antibody agarose
Manufactured by Merck Group
Sourced in Japan
The Anti-FLAG M2 antibody agarose is a resin-based affinity matrix used for the purification and detection of FLAG-tagged recombinant proteins. The antibody is covalently coupled to agarose beads, providing a robust and efficient platform for the isolation and enrichment of FLAG-tagged proteins from complex biological samples.
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3 protocols using anti flag m2 antibody agarose
1
Recombinant hResistin Protein Production
2
Purification and Characterization of Recombinant Human Resistin
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We produced hResistin in a eukaryotic cell line [6 (link)]. Briefly, the pcDNA5/FRT/TOPO TA vector containing C-terminal FLAG-tagged hResistin cDNA was integrated into the genome of the Flp-In™ T-REx™ 293 cell line in a Flp recombinase-dependent manner (Invitrogen, Carlsbad, CA). Production of recombinant (r) hResistin in T-REx 293 cells was induced by 1 μg/mL tetracycline. hResistin protein then was purified from the cell culture medium by anti-FLAG M2 antibody agarose (Sigma, St. Louis, MO) column chromatography. To determine the purity of eluted hResistin proteins, SDS-PAGE were employed using 4–20% Criterio Tris-HCl protein gel (#3450033, Bio-Rad, Hercules, CA) and Coomassie (#1610436, Bio-Rad) staining. We then stimulated 3T3-L1 embryonic fibroblasts (ATCC® CL-173™, ATCC, Manassas, VA) with hResistin at different doses for 10 minutes and lysed the cells with Laemmli sample buffer. We analyzed the cell lysates by immunoblotting with anti-phospho-Akt [2 (link), 18 (link), 20 (link)] (#4060, Cell Signaling Technology, Danvers, MA) and anti-GAPDH (G8795, Sigma-Aldrich) to determine the activity of the purified hResistin. Western blot analysis was performed as previously described [18 (link)]. The Trans-Blot Turbo Nitrocellulose Transfer Kit (#1704271, Bia-Rad) were used and protein bands were visualized by chemiluminescence (ECL; RPN2106, GE Healthcare, Marlborough, MA).
3
Purification and Immunization of Recombinant N. caninum Antigens
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Each recombinant BmNPV bacmid DNA injection into silkworm larvae and rearing silkworm larvae was performed according to the previous report (21) . Hemolymph was collected from silkworm larvae by cutting the prolegs, and 1-phenyl-2-thiourea was added into the collected hemolymph at 5 mM to prevent melanization. Collected hemolymph was centrifuged at 10000 × g for 15 min to remove hemocytes and debris, and its supernatant was used as the hemolymph sample.
To purify expressed recombinant N. caninum-antigens, 1 ml of anti-FLAG M2 antibody agarose (Sigma Aldrich Japan) was packed in an empty column and equilibrated with Tris-buffered saline (TBS; pH 7.5). Hemolymph was diluted 5-fold with TBS and loaded onto the anti-FLAG M2 antibody agarose column. The column was washed with 10 ml of TBS after loading the hemolymph and proteins were eluted with 8 ml of glycine-HCl buffer (pH 3.5). Every fraction of the 1 ml eluent was collected. caninum Nc-Liverpool was injected intraperitoneally into immunized mice and they were reared for 5 weeks. Blood and brains were collected and serum was prepared by centrifuging the blood at 1,000 × g.
To purify expressed recombinant N. caninum-antigens, 1 ml of anti-FLAG M2 antibody agarose (Sigma Aldrich Japan) was packed in an empty column and equilibrated with Tris-buffered saline (TBS; pH 7.5). Hemolymph was diluted 5-fold with TBS and loaded onto the anti-FLAG M2 antibody agarose column. The column was washed with 10 ml of TBS after loading the hemolymph and proteins were eluted with 8 ml of glycine-HCl buffer (pH 3.5). Every fraction of the 1 ml eluent was collected. caninum Nc-Liverpool was injected intraperitoneally into immunized mice and they were reared for 5 weeks. Blood and brains were collected and serum was prepared by centrifuging the blood at 1,000 × g.
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