The cpxR gene was cloned into the pET28a(+) plasmid (Novagen, Madison, WI, USA), and the recombinant proteins were expressed in E. coli BL21 (DE3) cells by addition of 1 mM isopropyl-β-d -thiogalactopyranoside. The purification of CpxR fusion protein was performed with a HisTrap high-performance column (GE Healthcare, Little Chalfont, Buckinghamshire, UK) as previously described [14 (link)]. The CpxR protein was phosphorylated with acetyl phosphate (Sigma, St. Louis, MO, USA) as previously described [51 (link)]. Then, EMSA were performed to determine the binding of phosphorylated CpxR (CpxR-P) to the hcp2B promoter. Briefly, the sequence of the hcp2B promoter region with or without the putative CpxR binding site was amplified and labeled with biotin. The biotin-labeled DNA probe (40 ng) was incubated with increasing concentrations of CpxR-P protein in EMSA binding buffer (10 mM Tris, 50 mM KCl, 5 mM MgCl2, 1 mM dithiothreitol, 0.1 mM MnCl2, 2.5% glycerol and 50 ng/μL poly[dI-dC]). After incubation for 30 min at room temperature, the reactions were subjected to electrophoresis and transferred to a nylon membrane. The biotin-labeled DNA was detected with a chemiluminescent substrate (Amersham Pharmacia Biotech). A competitive EMSA was performed by simultaneously incubating the biotin-labeled and unlabeled hcp2B promoter region with CpxR-P protein.
Acetyl phosphate
Manufactured by Merck Group
Sourced in United States
Acetyl phosphate is a compound used in biochemical and molecular biology research. It serves as a high-energy phosphate donor, participating in various enzymatic reactions. The core function of acetyl phosphate is to provide a source of phosphate for the transfer of energy and the modification of other molecules within laboratory experiments.
Automatically generated - may contain errors
Lab products found in correlation
Emsa probe biotin labeling kit, by Beyotime (3 mentions)
Chemiluminescent emsa kit, by Beyotime (3 mentions)
Qiaquick pcr purification kit, by Qiagen (2 mentions)
Nylon membrane, by Beyotime (2 mentions)
Lightshift chemiluminescent emsa kit, by Thermo Fisher Scientific (2 mentions)
Histrap high performance column, by GE Healthcare (1 mentions)
Chemiluminescent substrate, by Cytiva (1 mentions)
Carbamoyl phosphate, by Merck Group (1 mentions)
Quantity one image analysis software, by Bio-Rad (1 mentions)
Chemidoc xrs system, by Bio-Rad (1 mentions)
Perchloric acid pca, by Avantor (1 mentions)
Oxaloacetic acid, by Merck Group (1 mentions)
Citrate synthase, by Merck Group (1 mentions)
Monosodium phosphate, by Merck Group (1 mentions)
Dc protein assay kit, by Bio-Rad (1 mentions)
Sodium acetate, by Merck Group (1 mentions)
Hplc grade acetonitrile, by Avantor (1 mentions)
Dithiothreitol (dtt), by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Gemini Bio (1 mentions)
Tris hcl, by Merck Group (1 mentions)
24 protocols using acetyl phosphate
1
CpxR Binding to hcp2B Promoter by EMSA
2
Production and Phosphorylation of His6-PhoP Protein
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His6-PhoP protein was produced and purified as previously described (Gal-Mor et al., 2011 (link)) with some modifications (Cardenal-Muñoz and Ramos-Morales, 2013 (link)). For binding assays, S. enterica His6-PhoP was phosphorylated with acetyl phosphate as previously described (Tang et al., 2012 (link)) with modifications. Briefly, His6-PhoP was incubated in 20 μl of phosphorylation buffer (50 mM Tris-HCl pH 7.5, 50 mM KCl, 10 mM MgCl2) containing 10 mM acetyl phosphate (Sigma-Aldrich) for 1 h at 37°C.
3
Purification and Phosphorylation of PhoP
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Cultures of E. coli BL21 (DE3)/pCA24N:phoP were incubated at 37°C in LB broth medium supplied with corresponding antibiotics until the exponential phase. IPTG (isopropyl β-D-thiogalactoside) was added to produce a final concentration of 0.1 mM, and cultures were then incubated overnight with shaking at 16°C. Bacterial cells were harvested and then disrupted by sonication. The cell lysate was centrifuged at 10,000 × g for 30 min and the protein was isolated from the supernatant by nickel affinity chromatography. The purified His6-PhoP protein was dialyzed against 50 mM Tris–HCl buffer (pH 7.5). For EMSA assays, the His6-PhoP was phosphorylated as previously described (Tang et al., 2012 (link)) with modifications. Briefly, His6-PhoP was incubated in phosphorylation buffer (50 mM Tris–HCl [pH 7.5], 50 mM KCl, 10 mM MgCl2) containing 10 mM acetyl phosphate (Sigma-Aldrich) for 1 h at 37°C.
4
Phosphorylation Reactions of AbrA2, AbrA2N, and DrrBN
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Phosphorylation reactions were carried out with 2.5 µg of AbrA2, AbrA2N, or DrrBN protein, used as a control in the experiments [24] (link). The phosphorylation buffer was 50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 10 mM MgCl2 and the low-weight phosphor-donors used were 20 mM acetylphosphate (Sigma), 100 mM carbamoylphosphate (Sigma) and 150 mM phosphoramidate (obtained by chemical synthesis as in Sheridan et al. [25] ). The reactions were incubated at 37°C for 30 minutes, after which an SDS-PAGE loading buffer without DTT was added to stop the reaction and was loaded directly (without boiling) into an SDS-PAGE gel. Electrophoresis was performed at 4°C. The phosphorylated proteins were visualized with Phos-tag 300/460 Phosphoprotein Gel Stain (Perkin Elmer).
5
In Vitro Phosphorylation of UvrY Protein
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For in vitro phosphorylation, the purified UvrY-His6 protein was incubated with 100 mM acetyl-phosphate (Sigma) in a phosphorylation buffer containing 50 mM HEPES pH7.5, 100 mM NaCl and 10mM MgCl2. The reaction mixtures were incubated for 60 min at room temperature. To determine relative amount of UvrY-P formed, the reactions were fractionated on Phos-tagTM SDS-PAGE gels [1.0mm Protean 3 (Bio-Rad) minigels] of the following composition: 7.5% separating gel [7.5% (29:1) acrylamide: bis-acrylamide, 357mM Bis-Tris, pH 6.8, 100 μM Zn (NO3)2, and 50 μM Phos-tag reagent (Waco Pure Chemical Industries)] with a 4% stacking gel [4% (29:1) acrylamide:bis-acrylamide, 357 mM Bis-Tris, pH 6.8]. UvrY-P was resolved by electrophoresis at constant 150 V at 4°C for 70 min using modified MOPS running buffer (100 mM Tris, 100 mM MOPS, 0.5% SDS, and 5 mM NaHSO3). Gels were subsequently stained with Coomassie blue and the signals were imaged using a ChemiDoc XRS+ system (Bio-Rad) and quantified using Quantity One image analysis software (Bio-Rad).
6
Acetyl-CoA Synthesis and Quantification
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All solutions were prepared in Millipore water (Milli-Q system, Billerica, MA, USA). Aqueous solutions (72%) of perchloric acid (PCA) and HPLC grade acetonitrile were obtained from JT Baker (Phillipsburg, NJ, USA); the trilithium salt of acetyl-CoA, the sodium salt hydrate of CoA, sodium acetate, monosodium phosphate, 85% aqueous phosphoric acid, oxaloacetic acid, citrate synthase (from porcine heart; ammonium sulfate suspension; >1000 U/mg), acetyl phosphate, Tris HCl, and dithiothreitol (DTT) were obtained from Sigma Aldrich (Saint Louis, MO, USA); phosphotransacetylase (lyophilized preparation from Bacillus stearothermophilus, ~1000 U/mg solid) was obtained from Boehringer Mannheim (Ridgefield, CT, USA); Dulbecco Modified Eagle’s Medium (DMEM) was obtained from VWR Life Sciences (Radnor, PA, USA); fetal bovine serum (FBS) was obtained from Gemini Bio-Products (West Sacramento, CA, USA); and, the Bio-Rad DC™ protein assay kit was obtained from Bio-Rad (Hercules, CA, USA). Nylon membrane filters with a diameter of 47 mm and a pore size of 0.2 µm that were used for mobile phase filtering and degassing were obtained from Pall Life Science (Port Washington, NY, USA).
7
Reconstitution of E. coli Polar Lipids
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E. coli polar lipid extract (EcL) and 1,2-dioleoyl-sn-glycero-3-[(N-(5-amino-1-carboxypentyl)iminodiacetic acid)succinyl] (nickel salt) DGS-NTA, from Avanti Polar Lipids, Inc. (Alabaster, AL), were kept as 10–20 g/l stocks in chloroform solutions. Alexa Fluor 488 succinimidyl ester was from Molecular Probes/Invitrogen. Silica beads (nominal diameter ∼5 μm, 10.2% suspension in DI water solution) were from Bangs Laboratories, Inc. (Fishers, IN). Acetate kinase, acetyl phosphate and GTP were from Sigma. All reactants and salts were of analytical grade, from Merck. Ethanol was spectroscopic grade, also from Merck.
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E. coli polar lipid extract (EcL) and 1,2-dioleoyl-sn-glycero-3-[(N-(5-amino-1-carboxypentyl)iminodiacetic acid)succinyl] (nickel salt) DGS-NTA, from Avanti Polar Lipids, Inc. (Alabaster, AL), were kept as 10–20 g/l stocks in chloroform solutions. Alexa Fluor 488 succinimidyl ester was from Molecular Probes/Invitrogen. Silica beads (nominal diameter ∼5 μm, 10.2% suspension in DI water solution) were from Bangs Laboratories, Inc. (Fishers, IN). Acetate kinase, acetyl phosphate and GTP were from Sigma. All reactants and salts were of analytical grade, from Merck. Ethanol was spectroscopic grade, also from Merck.
8
Investigating Gene Regulation via EMSA
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The promoter regions of rpoE and pgaABCD genes were cloned into the T-vector pMD19-T to generate pMD19-T-rpoE and pMD19-T-pgaABCD. The promoter regions of rpoE and pgaABCD were then amplified via PCR from the plasmid using primers M13F-47 (FAM) and M13R-48, generating fluorescent FAM-labeled probes.
The recombinant protein CpxR was phosphorylated by acetyl phosphate (Sigma, USA) (Pogliano et al., 1997 (link)). Electrophoretic mobility shift assays (EMSAs) were carried out in 20 μL of reaction buffer (50 mM Tris-HCl (pH 8.0), 2.5 mM MgCl2, 100 mM KCl, 2 μg of salmon sperm DNA, 0.2 mM DTT, and 10% glycerol) that contained 40 ng of probe and varied quantities of CpxR or RpoE proteins. After incubation for 30 min at 30°C, the reaction liquid was loaded onto 4% non-denaturing PAGE gels in 0.5× TBE [5.4 g L−1 of Tris base, 2.75 g L−1 of boric acid, 2 mL L−1 of 0.5 M EDTA (pH 8.0)]. The resulting gel was photographed using an ImageQuant LAS 4000 mini system (GE Healthcare, USA).
The recombinant protein CpxR was phosphorylated by acetyl phosphate (Sigma, USA) (Pogliano et al., 1997 (link)). Electrophoretic mobility shift assays (EMSAs) were carried out in 20 μL of reaction buffer (50 mM Tris-HCl (pH 8.0), 2.5 mM MgCl2, 100 mM KCl, 2 μg of salmon sperm DNA, 0.2 mM DTT, and 10% glycerol) that contained 40 ng of probe and varied quantities of CpxR or RpoE proteins. After incubation for 30 min at 30°C, the reaction liquid was loaded onto 4% non-denaturing PAGE gels in 0.5× TBE [5.4 g L−1 of Tris base, 2.75 g L−1 of boric acid, 2 mL L−1 of 0.5 M EDTA (pH 8.0)]. The resulting gel was photographed using an ImageQuant LAS 4000 mini system (GE Healthcare, USA).
9
Acetylphosphate Activation of Response Regulators
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The response regulator (MbrC and VicR) proteins were activated by acetylphosphate (Sigma). An aliqot of the protein was incubated for 2 h at RT (room temperature) in reaction buffer (25 mM acetylphosphate, 50 mM Tris–HCl, 50 mM KCl, 10 mM MgCl2, 4 mM dithiothreitol) as described previously. Excess of acetylphosphate was removed by filtration and the protein was serially diluted in binding buffer (10 mM Tris, 1 mM EDTA, 100 mM KCl, 100 μM DTT, 5% vol/vol glycerol, 10 μg/ml BSA, pH 7.5). 0.5 pmol of target and competitor DNA was added to each reaction and incubated for 1 h at RT. The unrelated response regulator VicR which has a similar MW and pI as the response regulator MbrC under study was used as a negative control to rule out unspecific DNA binding. Four μl of the reaction mixture was applied on a 5% acrylamide gel run in Tris-borate-EDTA (TBE) buffer at pH 7.4. Gels were stained using SybrGold and visualized in a transilluminator (Alpha DigiDoc, Biorad) at 254 nm.
10
OmpR Binding to acrR and acrAB Promoters
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OmpR binding studies were performed using purified N-terminal His-tagged OmpR protein and DNA fragments containing the acrR and acrAB control regions. The acrR promoter fragment p222 used in the transcriptional fusion with gfp was employed in the EMSA. However, a new acrAB promoter fragment p225 was amplified for this purpose by PCR with primer pair FacAB1/RacAB225 (S1 Table ). OmpR-His6 synthesized in E. coli M15 was purified using Ni2+-NTA agarose as described previously [30 (link)]. The EMSA method was adapted from a previously published protocol [31 (link)]. For phosphorylation of OmpR, 20 mM acetyl phosphate (Sigma) was used. To confirm binding specificity, a 304-bp fragment of Y. enterocolitica Ye9 16S rDNA generated by PCR using primer pair 16SR1/16SR304 (S1 Table ) was included as a non-specific competitor in all binding reactions. The reaction mixtures were analyzed by electrophoresis on 6% non-denaturing polyacrylamide gels run in TBE buffer (Tris-borate-EDTA) and DNA bands were stained with SYBR Green EMSA nucleic acid gel stain (Invitrogen).
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