The sources of chemicals used in this study were as follows: Furylfuramide (AF-2; CAS No. 3688-53-7) and isopropyl β-D-thiogalactopyranoside (IPTG) were from Wako Pure Chemical Industries, Osaka, Japan; Methylmethane sulfonate (MMS; CAS No. 66–27-3) was purchased from Aldrich Chemical Co., Milwakukee, WI; AFB1 (CAS No. 1162–65-8) came from Sigma Chemicals, St. Louis, MO; 1,8-dinitropyrene (1,8-DNP; CAS No. 42397–65-9) was a gift from Dr. Naoki Miyata, National Institute of Health Sciences, Tokyo, Japan and azidoglycerol (AG; CAS No. 73018–99-2) synthesized as described previously [21 (link)] was kindly provided by Dr. T. Gichner, Institute of Experimental Botany, Academy of Sciences of the Czech Republic, Prague, Czech Republic. The test mutagens were dissolved in DMSO prior to use except MMS and AG which were dissolved in water. Rat liver 9000 x g supernatant fraction (S9) from animals pretreated with phenobarbital and 5,6-benzoflavone was purchased from Kikkoman, Chiba, Japan. All other chemicals were of analytical purity and have been purchased from local supplier (Wako Pure Chemical Industries, Osaka, Japan).
Isopropyl β d thiogalactopyranoside iptg
Manufactured by Fujifilm
Sourced in Japan
Isopropyl-β-d-thiogalactopyranoside (IPTG) is a synthetic chemical compound used in molecular biology and biotechnology laboratories. Its core function is to induce the expression of genes in bacteria that have been engineered to contain the lac operon. IPTG acts as an artificial inducer, triggering the transcription of target genes, allowing researchers to control and study gene expression.
Automatically generated - may contain errors
Lab products found in correlation
Glutathione sepharose column, by Cytiva (2 mentions)
Bugbuster protein extraction reagent, by Merck Group (2 mentions)
Methyl methanesulfonate mms, by Merck Group (1 mentions)
L homocysteine, by Merck Group (1 mentions)
L alanine, by Fujifilm (1 mentions)
L valine, by Fujifilm (1 mentions)
L methionine, by Fujifilm (1 mentions)
L serine, by Fujifilm (1 mentions)
Pgex 4t 1, by Cytiva (1 mentions)
Ampicillin, by Merck Group (1 mentions)
Lb medium, by BD (1 mentions)
Phosphoric acid, by Fujifilm (1 mentions)
Ammonia solution, by Fujifilm (1 mentions)
Ammonium iron 2 sulfate hexahydrate, by Fujifilm (1 mentions)
Glutathione sepharose column, by GE Healthcare (1 mentions)
Glycine, by Fujifilm (1 mentions)
Cucl2 2h2o, by Fujifilm (1 mentions)
Znso4 7h2o, by Fujifilm (1 mentions)
Potassium chloride (kcl), by Fujifilm (1 mentions)
Na2hpo4 12h2o, by Fujifilm (1 mentions)
9 protocols using isopropyl β d thiogalactopyranoside iptg
1
Genotoxicity Evaluating Diverse Chemicals
2
Biochemical Compounds Procurement Protocol
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NAD disodium salt, d -(–)-3-phosphoglyceric acid disodium salt, and l -homocysteine were purchased from Sigma-Aldrich Co., Ltd. (St. Louis, MO, USA). l -Alanine, l -valine, l -methionine, l -homoserine, l -serine, and N-Tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (TAPS), DTT, NADH, and isopropyl-β-d -thiogalactopyranoside (IPTG) were purchased from FUJIFILM Wako Pure Chemical Industries, Ltd. (Osaka, Japan).
3
Purification of WDR1 and CFL1 Proteins
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cDNA clones for CFL1 and WDR1 were separated using SEREX screening from the λZAP II phage cDNA library for the human esophageal carcinoma cell line, T.Tn (3 (link),4 (link),8 (link)), and human aortic endothelial cells (14 (link),15 (link)), respectively. Full-length cDNAs of WDR1 were recombined into pGEX-4T-1 (Cytiva). ECOSTM competent Escherichia coli BL-21 cells (Nippon Gene, Co., Ltd.) were transformed with prokaryotic expression plasmids, pGEX-4T-1, pGEX-4T-1-WDR1 and pGEX-4T-1-CFL1 (Cytiva), and then cultured for 3 h in a 200 ml Luria broth containing 0.1 mM isopropyl β-D-thiogalactopyranoside (IPTG; FUJIFILM Wako Pure Chemical Corporation). The cells were lysed by sonication in BugBuster Protein Extraction Reagent (Merck), and GST, GST-WDR1 and GST-CFL1 proteins were purified by affinity chromatography using glutathione-Sepharose columns (Cytiva), as previously described (16-18 (link)).
4
Recombinant Protein Expression in E. coli
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The plasmid of interest was transformed into E. coli strain BL21(DE3) and grown overnight as 100-ml preculture in LB medium (BD) with ampicillin (100 μg ml−1; Sigma-Aldrich) at 37°C with shaking at 180 rpm. The preculture was used to inoculate 2 to 8 liters of main culture that was grown at 37°C with shaking until the OD600 (optical density at 600 nm) reached ~0.8, after which the temperature was lowered to 20°C and protein expression was induced overnight with 0.4 mM isopropyl-β-d -thiogalactopyranoside (IPTG) (Wako). Cells were harvested by centrifugation, resuspended in lysis buffer [50 mM tris-HCl (pH 7.5), 10% glycerol, 0.1% Triton X-100], and stored at −80°C until further use.
5
Scalable Recombinant Protein Production
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Cultivation was performed at KNC Laboratories. For preculture, 100 µL of pFusionBM3‐WT/BL21 (DE3) glycerol stock was inoculated in 500 mL of modified LB liquid culture medium (animal‐derived material‐free) containing 25 µg/mL kanamycin sulfate and cultured at 25°C for 22 hours with shaking at 120 rpm.
1 L of precultured liquid was added to 150 L of modified 2 × YT medium (animal‐derived material‐free) containing 25 µg/mL kanamycin sulfate, 80 µg/mL 5‐aminolevulinic acid (Wako), 100 µM ammonium iron (II) sulfate hexahydrate (Wako), 250 µmol/L isopropyl β‐D‐thiogalactopyranoside (IPTG; Wako) and cultured at 20°C for 47 hours with ventilation rate of 75 L/min. 2 × YT medium was comprised of 1.6% (wt/vol) Difco select soytone, 1% (wt/vol) Bacto yeast extract and 0.5% (wt/vol) sodium chloride. pH in culture was maintained at pH 7.0 ± 0.1 using 25% (vol/vol) ammonia solution (Wako) and 2 mol/L phosphoric acid (Wako), and dissolved oxygen was maintained at DO 1.5 ± 0.5 ppm by stirring.
1 L of precultured liquid was added to 150 L of modified 2 × YT medium (animal‐derived material‐free) containing 25 µg/mL kanamycin sulfate, 80 µg/mL 5‐aminolevulinic acid (Wako), 100 µM ammonium iron (II) sulfate hexahydrate (Wako), 250 µmol/L isopropyl β‐D‐thiogalactopyranoside (IPTG; Wako) and cultured at 20°C for 47 hours with ventilation rate of 75 L/min. 2 × YT medium was comprised of 1.6% (wt/vol) Difco select soytone, 1% (wt/vol) Bacto yeast extract and 0.5% (wt/vol) sodium chloride. pH in culture was maintained at pH 7.0 ± 0.1 using 25% (vol/vol) ammonia solution (Wako) and 2 mol/L phosphoric acid (Wako), and dissolved oxygen was maintained at DO 1.5 ± 0.5 ppm by stirring.
6
Recombinant Production of PCSK9 Protein
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The full-length coding sequence of human PCSK9 gene (NCBI Accession number: NM_174936.3) was recombined into EcoRI/XhoI site of pGEX-4T-1. ECOS™ competent Escherichia coli JM-109 cells (Nippon Gene) were transformed with the eukaryotic expression plasmid pGEX-4T-1 or pGEX-4T-1-PCSK9 and then cultured for 3 h in 200 mL of Luria broth (LB) containing 0.1 mM isopropyl β-d -thiogalactopyranoside (IPTG; Wako Pure Chemicals, Osaka, Japan). Next, the cells were harvested, washed with phosphate-buffered saline, and lysed by sonication in BugBuster Protein Extraction Reagent (Merck Millipore, Darmstadt, Germany). Lysates were centrifuged at 15,000 g for 10 min at 4°C, and GST and GST-fused PCSK9 proteins were purified by affinity chromatography using glutathione–Sepharose columns (GE Healthcare Life Sciences) as described previously (34 (link)–36 (link)).
7
Purification and Characterization of Proteases
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Chemical reagents such as tris, glycine, CuCl2·2H2O, ZnSO4·7H2O, KCl, Na2HPO4·12H2O, NaCl, KH2PO4, EDTA·2Na, and isopropyl-β-d -thiogalactopyranoside (IPTG) were purchased from Wako Pure Chemical Industries Ltd., Osaka, Japan (Guaranteed Reagent). CaCl2.2H2O was purchased from Nacalai Tesque (Kyoto, Japan). The synthetic substrates such as Arg-pNA, Glu-pNA, Leu-pNA, Ala-pNA, and Phe-Leu-pNA were purchased from Peptide Institute Inc., Osaka, Japan. Tryptone and yeast extract were purchased from Becton-Dickinson and Company, NJ, USA.
8
Cloning and Inhibition Assay Protocol
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Oligonucleotide primers were synthesized by Life Technologies (Carlsbad, CA). TaKaRa Mighty Cloning Reagent Sets and a Ni-NTA agarose column were purchased from Life Technologies (Carlsbad, CA).
Restriction enzymes and Lambda HindIII DNA size markers were purchased from New England Biolabs, Inc. (Ipswich, MA). Relaxed pBR322 DNA was purchased from John Innes Enterprises Ltd (Norwich, UK).
Isopropyl-β-D-thiogalactopyranoside (IPTG) was purchased from Wako Pure Chemical Industries Ltd (Tokyo, Japan). E. coli BL21 (DE3) was purchased from Merck KGaA (Darmstadt, Germany).
For the inhibitory assay, three quinolones were used. Ciprofloxacin was purchased from LKT Laboratories Inc. (St Paul, MN). Norfloxacin and nalidixic acid were purchased from Wako Pure Chemical Industries Ltd. (Osaka, Japan).
Restriction enzymes and Lambda HindIII DNA size markers were purchased from New England Biolabs, Inc. (Ipswich, MA). Relaxed pBR322 DNA was purchased from John Innes Enterprises Ltd (Norwich, UK).
Isopropyl-β-D-thiogalactopyranoside (IPTG) was purchased from Wako Pure Chemical Industries Ltd (Tokyo, Japan). E. coli BL21 (DE3) was purchased from Merck KGaA (Darmstadt, Germany).
For the inhibitory assay, three quinolones were used. Ciprofloxacin was purchased from LKT Laboratories Inc. (St Paul, MN). Norfloxacin and nalidixic acid were purchased from Wako Pure Chemical Industries Ltd. (Osaka, Japan).
9
Purification of GST-fused Proteins
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ECOSTM competent Escherichia coli BL21-109 cells (Nippon Gene) were transformed with the eukaryotic expression plasmid, pGEX-4T-1 or pGEX-4T-1-FH, and then cultured for 3 h in 200 mL of Luria broth containing 0.1 mM isopropyl β-D-thiogalactopyranoside (IPTG; Wako Pure Chemicals, Osaka, Japan) [31 (link)]. The cells were then harvested, washed with phosphate-buffered saline, and lysed by sonication in BugBuster Protein Extraction Reagent (Novagen, San Diego, CA, USA). Lysates were centrifuged at 15,000× g for 10 min at 4 °C, and glutathione S-transferase (GST) and GST-fused FH proteins were purified using affinity chromatography with glutathione–Sepharose columns (Cytiva, Pittsburgh, PA, USA) as previously described [32 (link)].
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