The titanium dioxide, Luria–Bertani (LB) media, antibiotics (ampicillin and tetracycline), trypan blue solution (0.4%), Bioultra lyophilized proteinase K, and high-resolution agarose were obtained from Sigma-Aldrich Corp. (Milwaukee, WI, USA). The YOYO solution was purchased from Thermo Scientific (Waltham, MA, USA). The substrates for β-galactosidase (o-nitrophenyl-β-d -galactopyranoside) and alkaline phosphatase (p-nitrophenylphosphate) were purchased from Amresco (Slon, OH, USA). The Dulbecco’s modified Eagle medium (DMEM), Ham’s nutrient mixture F12 (F-12), fetal bovine serum (FBS), phosphate-buffered saline (PBS), trypsin EDTA solution, and antibiotics (penicillin–streptomycin mixture) were acquired from Gibco (Grand Island, NY, USA). Other reagents and solvents were obtained from commercial houses J.T. Baker (Phillipsburg, NJ, USA) or Merck (Kenilworth, NJ, USA).
O nitrophenyl β d galactopyranoside
Manufactured by Avantor
Sourced in United States
O-nitrophenyl-β-d-galactopyranoside is a chromogenic substrate used in biochemical assays. It is a synthetic analog of the natural substrate lactose, and its hydrolysis by β-galactosidase enzymes results in the release of a yellow colored product, o-nitrophenol, which can be detected and quantified spectrophotometrically.
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Lab products found in correlation
Titanium dioxide, by Merck Group (1 mentions)
High resolution agarose, by Merck Group (1 mentions)
Matchmaker gal4 two hybrid system 3 libraries user manual, by Takara Bio (1 mentions)
Yeast protocols handbook, by Takara Bio (1 mentions)
Glomax multi microplate multimode reader luminometer, by Promega (1 mentions)
Luciferase assay system, by Promega (1 mentions)
Polymyxin b, by Avantor (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Melittin, by Merck Group (1 mentions)
4 protocols using o nitrophenyl β d galactopyranoside
1
Standardized Reagents and Media for Cell Culture
2
Yeast Two-Hybrid Assay Protocol
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Y2H assays were performed as described previously (31 (link)). The pDEST-GBKT7–based bait vectors were transformed into the yeast strain Y187, and the yeast strain AH109 was transformed with pDEST-GADT7–based vectors. The fresh diploids by yeast mating were used to detect protein-protein interactions on selective triple dropout media (without Leu, Trp, and His) plus 1 mM 3-aminotriazole and quadruple dropout medium (without Leu, Trp, His, and Ade).
For yeast monohybrid assays (45 (link)), pDEST-GBKT7–based GAL4 DNA-BD fusion vectors were transformed into the yeast strain AH109 including several reporters (HIS3, MEL1, and lacZ) under distinct GAL4 upstream activating sequences as described in Matchmaker GAL4 Two-Hybrid System 3 & Libraries User Manual (Clontech). The transformants were grown on synthetic dropout agar medium lacking Trp and His (−WH) and detected on synthetic dropout/−WH/X-α-Gal (Biosynth). The liquid cultures of yeast cells were used to detect the lacZ expression in quantitative β-galactosidase assays with o-nitrophenyl-β-d -galactopyranoside (Amresco) performed according to the Yeast Protocols Handbook (Clontech).
For yeast monohybrid assays (45 (link)), pDEST-GBKT7–based GAL4 DNA-BD fusion vectors were transformed into the yeast strain AH109 including several reporters (HIS3, MEL1, and lacZ) under distinct GAL4 upstream activating sequences as described in Matchmaker GAL4 Two-Hybrid System 3 & Libraries User Manual (Clontech). The transformants were grown on synthetic dropout agar medium lacking Trp and His (−WH) and detected on synthetic dropout/−WH/X-α-Gal (Biosynth). The liquid cultures of yeast cells were used to detect the lacZ expression in quantitative β-galactosidase assays with o-nitrophenyl-β-
3
Luciferase Assay for Transfection Efficiency
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Luciferase Assay System (Promega) was used to measure luciferase (Luc) activity in cell lysates. Seventy-two hours after transfection, cells were washed with PBS and harvested using 50 μL of lysis buffer provided by the manufacturer. GloMaxMulti + Microplate Multimode Reader Luminometer (Promega) was used to measure luciferase activity. β-Galactosidase activity was assayed using 1 mg of o-nitrophenyl β-d-galactopyranoside (AmResco) as the substrate in buffer Z (40 mM NaH 2 PO 4 , 60 nM Ns 2 HPO 4 , M Schanton and others 594 Reproduction (2020) 160 591-602 10 nM KCl, 0.07% β-mercaptoethanol, 1 mM MgSO 4 ). Samples were incubated at 37°C until yellow staining. Enzymatic reaction was stopped with 75 μL of sodium carbonate. Color development was determined by absorption at 420 nm. The variation in transfection efficiency was corrected with this value. Luc results were calculated as the percentage of Luc activity per unit of β-galactosidase activity. For each data duplicates were performed.
4
Antimicrobial Peptide MDAP-2 Synthesis and Characterization
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Antimicrobial peptide MDAP-2 was synthesized by Shanghai Biotech Bioscience & Technology Co., Ltd.; Melittin was purchased from Sigma (USA); polymyxin B was purchased from Amresco (USA); O-nitrophenyl--β-D-galactopyranoside (ONPG) was purchased from Amresco (USA); N-phenyl-1-naphthylamine (NPN), bis-(1, 3-dibutylbarbituric acid) pentamethine oxonol (DiBAC4 (3)), propidium iodide (PI) and Triton X-100 were purchased from Sigma (USA).
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