Novel BET-inhibitor chemicals were identified by screening a DEL (WuXi AppTec, Shanghai, China) with the BD1s and BD2s from the BRD2, BRD3, and BRD4 proteins. These proteins were generated by amplifying the relevant sections of the BRD2–4-encoding genes, placing the sections into pET28a vectors (Novagen, Northumberland, UK), transforming Escherichia coli BL21(DE3) (Novagen) with each plasmid, and inducing protein expression by treatment with 0.2 mM isopropyl β-D-thiogalactopyranoside (Sigma, St. Louis, MO) for 16 h at 18°C. The bacteria were then collected, resuspended in lysis buffer (50 mM Tris, pH 8.2, with 300 mM NaCl and 20 mM imidazole), and sonicated. The lysates were centrifuged at 1,550×g for 1 h at 4°C, after which the supernatant was loaded onto a nickel affinity HisTrap HP column (GE Healthcare, Chicago, IL). After washing the column, BRD2, BRD3, or BRD4 was eluted with 50 mM Tris (pH 8.2) containing 300 mM NaCl and 500 mM imidazole.
Isopropyl β d thiogalactopyranoside
Manufactured by Merck Group
Sourced in United States, Germany, Cameroon
Isopropyl-β-D-thiogalactopyranoside is a synthetic organic compound used as an inducer in the expression of recombinant proteins in bacterial cultures. It is a lactose analog that binds to and inactivates the lac repressor, allowing transcription of the target gene to occur.
Automatically generated - may contain errors
Lab products found in correlation
Dithiothreitol (dtt), by Merck Group (13 mentions)
Imidazole, by Merck Group (13 mentions)
Ampicillin, by Merck Group (12 mentions)
Kanamycin, by Merck Group (11 mentions)
Bovine serum albumin (bsa), by Merck Group (10 mentions)
Triton x 100, by Merck Group (7 mentions)
Glycerol, by Merck Group (7 mentions)
Sodium dodecyl sulfate (sds), by Merck Group (6 mentions)
Oxidized glutathione, by Merck Group (6 mentions)
Hemin, by Merck Group (6 mentions)
Urea, by Merck Group (5 mentions)
Lb medium, by Merck Group (5 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (5 mentions)
Guanidine hydrochloride, by Merck Group (4 mentions)
Citric acid, by Merck Group (4 mentions)
Veratryl alcohol, by Merck Group (4 mentions)
Restriction endonuclease, by New England Biolabs (4 mentions)
Chloramphenicol, by Merck Group (4 mentions)
β mercaptoethanol, by Merck Group (4 mentions)
Histrap hp column, by GE Healthcare (3 mentions)
119 protocols using isopropyl β d thiogalactopyranoside
1
Identification of Novel BET Inhibitors
2
Cloning and Expression of STIM2-UI Protein
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Human STIM2 cDNA was obtained from Addgene (Cambridge, MA, USA, #18862). To prepare the cDNA for the STIM2-UI region, oligonucleotide primers were designed on the basis of human STIM2 (GenBank accession number: NM_ AF328905.1) (Table 2 , bottom panel). With these primers, PCR was performed (30 cycles at 95 °C for 45 s, 63 °C for 45 s, and 68 °C for 90 s). The PCR fragments were sub-cloned into pGEX-4T-1 vector with EcoR 1 and Sal I enzyme sites. The sequence of the construct was confirmed (ABI Prism 3700 DNA Sequencer, Applied Biosystems, ThermoFisher Scientific, Waltham, MA, USA). GST-fused STIM2-UI protein was expressed in E. coli (DH5α) using 0.1 mM isopropyl-β-D-thiogalactopyranoside (Sigma-Aldrich, St. Louis, MO, USA), as previously described79 (link),80 (link).
3
Purification and Analysis of AroH
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The plasmid AroHhis, which contains the entire AroH cDNA along with a C-terminal 6xHis tag cloned in the BamHI/NotI sites of a pET22b prokaryotic expression vector, was purchased from GenScript (New Jersey, USA). PLP, L-tryptophan, L-tyrosine, L-phenylalanine, α-aminoadipate, α-ketoglutarate, α-ketoadipate, L-glutamate dehydrogenase from bovine liver (GDH), 3-acetylpyridine adenine dinucleotide (APAD+), L-lactic dehydrogenase (LDH), and isopropyl-β-D-thiogalactopyranoside, were purchased from Sigma. All other chemicals were of the highest purity available.
4
Preparation of GST-Tagged CASQ1 Constructs
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cDNA of mouse CASQ1 was obtained from OriGene Technologies, Inc. (Rockville, MD, USA, #MR206274). To prepare cDNA for GST-tagged full-length CASQ1 (GST-CASQ1) or CASQ1 regions, oligonucleotide primers were designed based on mouse CASQ1 (GenBank accession number: NM_009813) (Supplementary Tables S1 and S2 ). With the primers, PCR was performed (30 cycles at 95 °C for 45 s, 63 °C for 45 s, and 68 °C for 90 s). The PCR products were subcloned into the pGEX-4T-1 vector. GST-CASQ1 or GST-CASQ1 regions were expressed in E. coli (DH5α) using 0.1 mM isopropyl-β-D-thiogalactopyranoside (Sigma–Aldrich, St. Louis, MO, USA), as previously described [22 (link),36 (link),37 (link)]. For expressing the full-length wild-type CASQ1 (WT CASQ1) in mouse primary skeletal myotubes, full-length CASQ1 in the pGEX-4T-1 vector was subcloned into the pCMS-RFP vector using EcoR I and Not I enzyme sites. For the C1 region, the PCR products using primers in Supplementary Table S3 were subcloned into the pCMS-RFP vector.
5
DARPP-32 Bacterial Expression and Purification
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cDNA encoding human DARPP-32 WT and the DARPP-32 T153A point mutant were cloned into the bacterial expression vector pGEX-4T-1 (GE Healthcare, Logan, Utah, USA) and transformed into Escherichia coli strain BL21(DE3) (Novagen, Darmstadt, Germany), respectively. For the expression of DARPP-32 WT and DARPP-32 T153A, transformed cells were grown in LB medium at 37 °C until an OD600 of 0.5 was reached. Protein expression was then induced by the addition of 0.5 mm isopropyl-β-d -thiogalactopyranoside (Sigma-Aldrich, St Louis, MO, USA) for 5 h at 28 °C. The recombinant proteins expressed were purified using GST•Bind Agarose Resin (Elpis Biotech) according to manufacturer’s instructions.
6
Recombinant β-glucosidase expression in E. coli
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The recombinant pGEX-4T-1 vector containing S. griseus β-glucosidase gene (GST-tagged) in E. coli BL21 (DE3) was developed in a previous study [14 (link)]. Ampicillin, glycerol, Isopropyl-β-D-thiogalactopyranoside (IPTG), LB broth, p-nitrophenyl-β-D-glucopyranoside (pNPG), p-nitrophenol (pNP), cellobiose, fructose sucrose, tryptone, yeast extract, beef extract, CaCl2, DTT, KOH, MgCl2, (NH4)2S4, ZnSO4, Triton X-100, M PMSF, Lysozyme, Bradford reagent, and Glutathione Sepharose 4B resin were purchased from Sigma Aldrich (Ireland).
7
Affinity Purification of Recombinant Proteins
8
Recombinant PbTIP Protein Expression
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For the expression of PbTIP, a segment of it encoding 204–335 amino acids was amplified from P. berghei genomic DNA with forward primer (3′-sequence-5′) and reverse primer (3′-sequence-5′). Using restriction enzymes BamHI and XhoI, rPbTIP was cloned into the expression vector pET32a (+) (Novagen, Darmstadt, Germany). The recombinant plasmid was transformed into Escherichia coli BL-21 (Novagen, Darmstadt, Germany). His-tagged rPbTIP was expressed at 20°C for 12 hr with 1 mmol isopropyl-β-D-thiogalactopyranoside (Sigma-Aldrich, St. Louis, Missouri, USA). Ultrafiltration was performed to remove the endotoxin from the bacterially produced protein (endotoxin<1 Eu/μg). The soluble rPbTIP was purified by Ni-NTA His-Bind Superflow (Invitrogen, Carlsbad, California, USA) in accordance to the manufacturer’s instructions. Purified rPbTIP was dialyzed with phosphate buffered saline (PBS) for 4 hr at 4°C. Then the purified recombinant PbTIP was analyzed on 10% SDS-PAGE gel.
9
Expression and Characterization of NrdR Variants
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Overnight cultures of E. coli BL21(DE3) bearing pET30a(+)::nrdR, pET30a(+)::nrdRΔNΔC, pET30a(+)::ScoNrdR_D15A, pET30a(+)::ScoNrdR_R17A, and pET30a(+)::ScoNrdR_D15A/R17A were diluted to an absorbance at 600 nm of 0.1 in LB (Luria-Bertani) liquid medium, containing kanamycin (50 μg/ml) and shaken vigorously at 37 °C. At an absorbance of 0.8 at 600 nm, isopropyl-β-d -thiogalactopyranoside (Sigma) was added to a final concentration of 0.4 mM; Zn(CH3CO2)2 was added to 0.1 mM to all cultures except culture bearing pET30a(+)::nrdRΔNΔC. The cells were grown overnight under vigorous shaking at 20 °C and harvested by centrifugation. The cell pellet was stored at −80 °C.
10
Preparation of Reagents for Biochemical Assays
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Chemicals and solvents (acrylamide, N,N′-methylenebisacrylamide, DTT, urea, glycerol, Hepes, isopropyl-β-d -thiogalactopyranoside, imidazole, Tris, NaCl, KCl, NaOH, EDTA, HCl, SDS, Coomassie Brilliant Blue G-250 and divalent metal salts CoCl2, MgCl2, MnCl2, ZnCl2, and NiSO4) were purchased from Sigma-Aldrich and Panreac Química SLU and used without further purification. dNTPs were bought from Biosan. All the solutions were prepared using double-distilled water.
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