The ORF corresponding to RodZ (ORF wcw_0755) was amplified by PCR using the primers RodZ_fwd and RodZ_rev and cloned into the pET200 vector (Life technologies) in E. coli. This vector allows the expression of a N-terminal tagged version of the protein under a isopropyl-β-D -thiogalactopyranoside (IPTG) inducible promoter. Protein was expressed with 1 mM IPTG (MP Biomedicals, Santa Ana, CA) during 4 h and purified in presence of 8 M urea. Bacteria were lysed using Fastbreak Lysis buffer (Promega). Lysate was then incubated with MagneHis Ni particles beads and beads were washed with binding/wash buffer (Promega). The purified protein was then eluted using 500 mM imidazole (Sigma-Aldrich)56 (link). The purified protein was then used to immunize mice (Eurogentec, Seraing, Belgium).
Magnehis ni particles
Manufactured by Promega
Sourced in United States
MagneHis Ni-Particles are paramagnetic beads coated with a nickel-charged surface. They are designed for the rapid and efficient purification of histidine-tagged proteins from biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Protease inhibitor, by Roche (2 mentions)
Pepsin, by Roche (2 mentions)
Qstar pulsar, by Thermo Fisher Scientific (2 mentions)
Imidazole, by Merck Group (1 mentions)
Isopropyl β d 1 thiogalactopyranoside (iptg), by MP Biomedicals (1 mentions)
Bl21 de3 plyss e coli cells, by Thermo Fisher Scientific (1 mentions)
Pwo dna polymerase, by Roche (1 mentions)
In fusion hd cloning kit, by Takara Bio (1 mentions)
Pet28b, by Merck Group (1 mentions)
Analyst qs software, by Thermo Fisher Scientific (1 mentions)
Vent dna polymerase, by New England Biolabs (1 mentions)
Alkaline phosphatase labeled anti mouse igg, by Promega (1 mentions)
T4 rna ligase, by Takara Bio (1 mentions)
T7 rna polymerase, by New England Biolabs (1 mentions)
Bca assay, by Thermo Fisher Scientific (1 mentions)
12 protocols using magnehis ni particles
1
Heterologous Expression and Purification of RodZ Protein
2
Enzymatic Glycosylation Assay Protocol
3
Murine His-tag NAGK Purification
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The murine His-tag NAGK clones in pRSET vectors were introduced into E. coli BL21 (DE3)-pLysS cells (Invitrogen) and chloramphenicol/ampicillin-resistant transformants were selected. Cells were grown to an OD600 of 0.5, induced with 1.0 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) for 3 h, and stored at −70°C. For protein purification, cells were thawed, sonicated, and enzymes were purified using a Ni-nitrilotriacetic acid column (MagneHis™ Ni-Particles) (Promega; USA) by following the manufacturer’s instructions.
4
CCHFV/HAZV particle co-capture with LDL-R or CD81
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CCHF tecVLPs or WT CCHFV or HAZV particles were incubated with 5μg of soluble LDL-R (2148-LD-025/CF, R&D systems), or CD81-LEL for 1 h at room temperature. Then 30uL of MagneHis Ni-Particles (Promega) were added and the samples were processed according to manufacturer’s instructions. For the elution, beads were resuspended in TriReagent and the supernatant was transferred into a new tube for RNA extraction, following manufacturer’s protocol before determination of the level of co-captured CCHFV or HAZV RNAs by RT-qPCR (see below).
5
Recombinant Protein Purification Procedure
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The ORFs of SdCPR and SdC4H were amplified using Pwo DNA polymerase (Roche). The PCR products were inserted into the expression vector pET28b (Merck Millipore, Burlington, MA, USA) using an In-fusion HD Cloning Kit (Takara Bio Inc.). E. coli BL21 (DE3) cells harboring the expression vector were grown overnight in LB medium with 50 μg mL−1 kanamycin and 1% glucose at 37 °C in a shaking incubator, then diluted 1:25 into fresh LB medium supplemented with 50 μg mL−1 kanamycin. Cells were grown at 37 °C at 200 rpm until absorbance at 600 nm reached 0.4 − 0.6, and then 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) was added. The culture was shaking at 200 rpm at 25 °C overnight for protein expression. The bacterial cells were collected by centrifugation at 3000 rpm for 5 min at 4 °C and washed twice with 4 °C wash buffer (10 mM Tris–HCl [pH 7.5], 150 mM NaCl). Then, the washed cell pellet was suspended in the BugBuster Protein Extraction Reagent (Novagen-Merck Millipore) and His-tag recombinant proteins were purified from the supernatant using MagneHis Ni-Particles (Promega) with elution buffer containing 1 M imidazole.
6
Syt1 Pulldown from Mouse Brain Lysates
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Syt1 tagged with V5 and His was overexpressed in PS DKO MEF cells. The protein was extracted from the cells using a lysis buffer containing 50 mM HEPES, 100 mM NaCl, protease inhibitors (Roche, Indianapolis, Indiana) and 1% TX-100, and was immobilized on the MagneHis Ni-Particles (Promega, Madison, WI) by 2-min incubation in the presence of 20 mM imidazole to reduce non-specific binding. Subsequently, 30 μl of the Syt1-V5-His bound beads were added to the mouse brain lysates containing 2 mg of total protein and prepared in a buffer containing 50 mM HEPES, 100 mM NaCl, protease inhibitors (Roche, Indianapolis, Indiana), 1% CHAPS, 20 mM imidazole, 2 mM CaCl2. 100 μg of PS1-LNT or scramble, as a negative control, peptides were added to the samples. Additional negative controls included Syt1-V5-His bound beads incubated with the lysis buffer only, or empty beads incubated with the mouse brain lysates. The mixtures were incubated overnight at 4 °C with end-over-end rotation. Then, the beads were washed 3 times with the 1% CHAPS-based lysis buffer, and the attached protein was eluted with the elution buffer provided with the MagneHis Ni-Particles, containing 500 mM imidazole.
7
In vitro Kinase Assay of EB2
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EB2 proteins tagged with His at the C terminus were expressed in Rosetta2 (DE3) pLysS and purified with MagneHis Ni-Particles (Promega). Purified EB2, histone H1 (as a positive control of CDK1, New England Biolabs) and histone H3 (as a positive control of Aurora B, New England Biolabs) proteins were incubated with active CDK1-Cyclin B (Millipore) and/or active Aurora B (Abcam) in kinase buffer. In vitro kinase assays were carried out in 30 μl of reaction buffer (50 mM Tris-HCl, 10 mM MgCl2, 1 mM EGTA, 2 mM dithiothreitol, 0.01% Brij 35, 50 μM cold ATP, 5 μCi [γ-32P]-ATP and 1 μg of purified EB2 proteins pH 7.5) containing 50 ng of CDK1-Cyclin B or 50 ng of Aurora B. Reactions were incubated at 30 °C for 30 min. Reaction samples were terminated by adding 4 × Laemmli buffer, boiled and subjected to SDS–PAGE followed by autoradiography.
8
Hydrogen-Deuterium Exchange Mass Spectrometry
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Native His6-MDH (2 µM and 400 µl), thermally aggregated His6-MDH, or sHsp/His6–MDH complexes (both formed for 30 min at 47°C) in buffer A (50 mM Hepes, pH 7.6, 50 mM KCl, 5 mM MgCl2, and 2 mM DTT) were incubated with 50 µl MagneHis Ni particles (Promega) for 15 min at RT. His6-MDH (aggregated or sHsp-bound) was isolated by placing the reaction in a magnetic rack. The supernatant was subsequently removed, and the beads were washed once with buffer A. D2O-based buffer A was added to initiate amide proton–deuteron exchange. After 30 s, the exchange reaction was quenched by adding ice-cold low-pH quench buffer (500 mM K-phosphate buffer, pH 2.2) containing pepsin (25 µg/ml; Roche). Protein was digested from the Ni particles for 1 min on ice. Then, quenched, digested samples were injected into the HPLC setup with online peptic digest and analyzed on an electrospray ionization quadrupole time-of-flight mass spectrometer (QSTAR Pulsar; Applied Biosystems) as previously described (Rist et al., 2003 (link)). Analysis of deuteron incorporation into peptides was performed by using AnalystQS software (MDS SCIEX; Applied Biosystems). The assignment of the isotope peaks and the selection of the peptides presented were done manually. Analysis of bimodal isotope distributions was performed as described previously (Ungelenk et al., 2016 (link)).
9
Probing Protein Aggregation by HDX-MS
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Native His6-MDH (2 μM, 400 μl), thermally aggregated His6-MDH or sHsp/His6-MDH complexes (both formed for 30 min at 47 °C) in buffer C (50 mM HEPES pH 7.6, 50 mM KCl, 5 mM MgCl2, 2 mM DTT) were incubated with 50 μl MagneHis Ni-Particles (Promega) for 15 min at room temperature. His6-MDH (aggregated or sHsp-bound) was isolated by placing the reaction in a magnetic reack. The supernatant was subsequently removed and the beads were washed once with buffer C. D2O-based buffer C was added to initiate amide proton-deuteron exchange. After 30 s the exchange reaction was quenched by adding ice-cold low pH quench buffer (500 mM K-phosphate buffer, pH 2.2) containing pepsin (25 μg ml−1, Roche). Protein was digested from the Ni-Particles for 1 min on ice. Quenched, digested samples were injected into the HPLC setup, with online peptic digest, and analysed on an electrospray ionization quadrupole time-of-flight mass spectrometer (QSTAR Pulsar, Applied Biosystems) as described in ref. 53 (link). Analysis of deuteron incorporation into peptides was performed by using AnalystQS software (Applied Biosystems/MDS SCIEX, Germany). The assignment of the isotope peaks and the selection of the peptides presented were done manually.
10
Streptavidin Mutant Binding Assay
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The DNAs of wild type and Tyr83 and Trp120 mutant streptavidin and DNA of the tRNA having four-base anticodon and BODIPY-FL-aminophenylalanyl-pdCpA were obtained from laboratory of Professor Hohsaka of Japan Advanced Institute of Science and Technology (JAIST). Alkaline phosphatase-labeled anti-mouse IgG and Magne His Ni-particles were obtained from Promega. Polymerase chain reaction (PCR) enzyme (Vent DNA polymerase) and T7 RNA polymerase were purchased from New England Biolabs. T4 RNA ligase was from Takara Bio. Anti-T7-tag antibody was obtained from Novagen. Biotin-(AC5)2-hydrazide was purchased from DOJINDO.
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