6xHis-tagged GFP was expressed in BL21-CodonPlus cells by IPTG induction at 37°C for 4 h. The fusion protein was subsequently purified by immobilized metal affinity chromatography using Ni-bound Sepharose. Approximately 1 mg of purified protein was used for rabbit immunization. Additional GFP protein was covalently coupled to Affi-gel 10 resin (Bio-Rad) and used for affinity purification of anti-GFP polyclonal antibodies from the sera.
Affi gel 10 resin
Manufactured by Bio-Rad
Sourced in United States
Affi-Gel 10 resin is a hydrophilic, activated agarose-based gel developed for the purification of proteins and other biomolecules. It features an N-hydroxysuccinimide (NHS) ester group that can be used to covalently couple ligands, enabling efficient and reversible binding of target molecules.
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Lab products found in correlation
Affi gel 10, by Bio-Rad (2 mentions)
Dye binding assay, by Bio-Rad (1 mentions)
Syntaxin 4, by Santa Cruz Biotechnology (1 mentions)
Complete protease inhibitor, by Roche (1 mentions)
Bradford assay, by Bio-Rad (1 mentions)
Ab818, by Abcam (1 mentions)
Gtx129674, by GeneTex (1 mentions)
Rabbit anti ha, by Cell Signaling Technology (1 mentions)
Glutathione agarose, by Thermo Fisher Scientific (1 mentions)
Bovine trypsin, by Merck Group (1 mentions)
Disuccinimidyl suberate, by Merck Group (1 mentions)
Benzyloxycarbonyl gly pro arg p nitroanilide z gpr pna, by Merck Group (1 mentions)
Synthetic oligonucleotides, by Integrated DNA Technologies (1 mentions)
T4 ligase, by New England Biolabs (1 mentions)
Q5 polymerase, by New England Biolabs (1 mentions)
17 protocols using affi gel 10 resin
1
Purification and Immunization of 6xHis-GFP
2
Immobilization of Compound A on Affi-Gel 10
3
Purification of Anti-Asp1 Antibody
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Rabbit immunization with purified Asp1-(1–364) and preparation of antiserum were performed by Pocono Hills Rabbit Farm and Laboratory (Canadensis, PA) according to their Mighty Quick Protocol. Anti-Asp1 antibody was purified from rabbit serum by affinity chromatography as follows. Purified Asp1 kinase (4.5 mg) was dialyzed against coupling buffer (100 mM HEPES, pH 6.5, 500 mM NaCl, 5% glycerol) and then coupled to 4 mL of Affigel-10 resin (Bio-Rad) during overnight incubation at 4°C. The resin was washed serially with 100 mM Tris-HCl, pH 7.5; 200 mM glycine, pH 2.6; 1 M Tris-HCl, pH 7.5; and 20 mM Tris-HCl, pH 7.5, 150 mM NaCl. Asp1 kinase-coupled resin was then mixed with 8 mL of rabbit immune serum (adjusted to 10 mM Tris-HCl, pH 7.5) for 2 h at room temperature on a nutator. The resin was poured into a column and washed thoroughly with 10 mM Tris-HCl, pH 7.5, 150 mM NaCl until no further protein eluted, as gauged by Bio-Rad dye-binding assay of wash fractions. Bound antibodies were eluted with 200 mM glycine, pH 2.6 while collecting fractions (1 mL) in tubes containing 100 μL of 1 M Tris-HCl, pH 7.5 to adjust the pH. Protein-containing eluate fractions were pooled, dialyzed against buffer containing 10 mM Tris-HCl, pH 7.5, 150 mM NaCl, and concentrated to 0.36 mg/mL.
4
Immobilizing DIF-1-NH2 onto Affi-Gel 10
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DIF-1-NH2 was coupled to Affi-Gel 10 resin (Bio-Rad, Hercules, CA, USA) at 4 °C to produce DIF–Affi-Gel 10 (DIF beads) (Figure 1 B) according to the manufacturer’s instructions, as previously described [18 (link)]. The DIF beads were washed well with 0.1 M phosphate-buffered saline and kept in EtOH at 4 °C until use.
5
Monoclonal Antibodies for Complement and Cell Markers
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Commercially available monoclonal antibodies were directed against a neoepitope of the terminal complement complex C5b-9 (M0777/aE11; DAKO, Carpinteria, CA), human vitronectin (MAB1945; Chemicon, Temecula, CA), human CD46 (#555948; BD Biosciences, San Jose, CA), and syntaxin-4 (SC-101301; Santa Cruz Biotechnology, Santa Cruz, CA). The HRGR-DE7 antibody, which is directed against the identical carboxyl termini of human RGR and RGR-d, and an RGR-d-specific antibody DE21 were produced as described previously. Briefly, rabbit antisera were generated by Cocalico Biologicals, Inc. (Reamstown, PA) by immunization with synthetic peptides conjugated to keyhole limpet hemocyanin. The polyclonal antibodies were purified from antisera by affinity-binding to immobilized peptide attached to Affi-Gel 10 resin (Bio-Rad, Hercules, CA). DE21 was shown to bind recombinant RGR-d protein specifically without binding to full-length RGR. The DE21 antibody is directed against the peptide sequence (GKSGHLQVPALIAK) that corresponds to the sequence of human RGR-d at the splice junction of exons 5 and 7.
6
Polyclonal Anti-TbITPA Antibody Production
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To obtain a polyclonal anti-TbITPA antibody, a rabbit was immunized using soluble purified recombinant TbITPA. Before injecting the antigen into the rabbit, 500 μg of pure protein diluted in PBS was mixed with Freund’s adjuvant (1:1 ratio). Antibody-containing serum was collected and affinity-purified using pure recombinant protein coupled to Affi-Gel 10 resin (BioRad) following the manufacturer’s instructions.
7
Immunoprecipitation of Influenza PB2 Protein
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ICC was performed as previously published [17 (link)] with the following modifications. Anti-FLAG clone 1/27 was coupled to Affi-Gel 10 resin following the manufacturer’s instructions (Bio-Rad). A549 cells were inoculated at an MOI of 0.2 with S009 PB2-FLAG or S009 PB2-627K-FLAG in a 10-cm dish, 5 dishes per replicate, 3 biological replicates. Infections were allowed to proceed for 24 h. Cells were combined and lysed in 1 ml co-IP buffer (50 mM Tris (pH 7.4), 150 mM NaCl, 0.5% NP-40, 1X cOmplete protease inhibitor (Roche)) and divided into equivalent fractions for ICC. Lysates were incubated by rocking for 3 h at 4°C with free competing antibody (anti-FLAG 1/27) where applicable, then immunoprecipitated for 16 h with 1/27 antibody coupled to Affi-Gel 10 resin. After immunoprecipitation, samples were washed four times with co-IP buffer and eluted in Laemmli buffer at 70°C for 10 min. Samples were then transferred to new tubes and boiled for 10 min with 0.1 M DTT. Approximately 10% of the immunoprecipitates were separated by SDS-PAGE and analyzed by western blotting using 1/54 anti-FLAG antibody or silver stained.
8
Affinity Purification of Antibodies
9
Immobilization of Compound A on Affi-Gel 10
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The inhibitor SK&F 93505 – (5R)-6-(4-aminophenyl)-5-methyl-4,5-dihydropyridazin-3(2H)-one – was synthesized as described56 . Compound A was prepared from SK&F 93505 as described39 (link). Compound A was linked to Affi-Gel 10 resin (BioRad) following the manufacturer’s protocol. Briefly, Affi-Gel 10 resin (6 mL) was loaded into a 1.5 × 10 cm fritted column and washed with 2 column volumes (CV) isopropanol, followed by 2 CV dimethylsulfoxide (DMSO). The resin was transferred to a 15 mL Falcon tube. Compound A was dissolved in DMSO to 100 mg/mL and 150 μL of this solution were added to the resin. The derivatization reaction proceeded for 2.5 h at room temperature with nutation. Excess sites on the resin were blocked by addition of a slight excess of ethanolamine (7 μL) and incubation at room temperature for 15 min. The resin was returned to the column and washed with several CV of isopropanol. Compound A was stored for extended periods in DMSO at -20 °C and the column was stored in isopropanol at 4 °C.
10
Rabbit Polyclonal Antibodies against RGR Protein
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We produced and authenticated rabbit polyclonal antipeptide antibodies against RGR. Synthetic peptides, and peptides conjugated to keyhole limpet hemocyanin for immunization, were obtained from the Caltech Biopolymer Synthesis Center (Caltech, Pasadena, CA). Rabbit antisera were generated by Cocalico Biologicals, Inc. (Reamstown, PA). The antibodies were purified with affinity chromatography using peptide immobilized to Affi-Gel 10 resin (Bio-Rad Laboratories, Hercules, CA). The DE15 antibody is directed against a peptide sequence (SSLLRRWPHGSEGC) that is partly conserved in RGR across several different species. The DE1 antibody was generated against a synthetic peptide (CLSPQRREHSREQ) that corresponds to the carboxyl terminus of bovine RGR [5 (link)]. The RGR-d-specific antibody, DE21, was generated against a synthetic peptide (GKSGHLQVPALIAK) that corresponds to the unique sequence of human RGR-d at the splice junction of exons 5 and 7 [17 (link)]. DE21 immunoreactivity was validated by the ability of the antibody to specifically bind recombinant human RGR-d protein [17 (link)]. The cone opsin antibodies, OPN1MW/LW (sc-22117) and OPN1SW (sc-14363), were obtained from Santa Cruz Biotechnology, Inc. (Dallas, TX).
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