The COS-7 cells were grown to 60–70% confluency on 2 × 15 cm dishes. Forty-eight hours post-transfection, the cells were lysed on ice for 30 min with PBS + 1%TridonX-100 + 1%NP-40 + 1%PMSF (DMSO free), pH = 7.5, then freeze-thawed 5 times, and ultrasonicated for 10 cycles. WT and mutant NADSYN1 protein was purified using HisPur™ Ni-NTA Purification Kit following the manufacturer’s instructions (88227, Thermo Fisher Scientific, Hudson, NH, USA). The equilibration, wash, and elution buffers were 10 mM, 25 mM, and 500 mM imidazole respectively. The resin beds were incubated with protein extract for 30 min on an end-overend rotor at 4 °C. The eluted proteins were quantified by BCA protein assay kit (Sigma-Aldrich, Darmstadt, Germany).
Hispur ni nta purification kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The HisPur™ Ni-NTA Purification Kit is a laboratory product designed for the purification of His-tagged proteins. It contains Ni-NTA agarose resin, buffers, and other necessary components for the process of affinity chromatography.
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Lab products found in correlation
Bca protein assay kit, by Merck Group (1 mentions)
Quickchange 2 site directed mutagenesis kit, by Agilent Technologies (1 mentions)
Hek293 cells, by Merck Group (1 mentions)
Bac to bac baculoviral expression system, by Thermo Fisher Scientific (1 mentions)
Protease inhibitor, by Roche (1 mentions)
Ampicillin, by Merck Group (1 mentions)
Kanamycin, by Merck Group (1 mentions)
Chloramphenicol, by Merck Group (1 mentions)
Isopropyl β d 1 thiogalactopyranoside (iptg), by AppliChem (1 mentions)
Rosetta 2 de3 cells, by Merck Group (1 mentions)
Glutathione sepharose 4b, by GE Healthcare (1 mentions)
Sonicator, by Bandelin (1 mentions)
Protein a g agarose, by Thermo Fisher Scientific (1 mentions)
Catch and release v2.0 reversible immunoprecipitation system, by Merck Group (1 mentions)
Lipofectamine 3000 transfection kit, by Thermo Fisher Scientific (1 mentions)
Amicon ultra 4 centrifugal filter device, by Merck Group (1 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
9 protocols using hispur ni nta purification kit
1
Purification of Recombinant NADSYN1 Proteins
2
Recombinant Factor B Mutagenesis
3
In Vitro Translation Silencing Assay
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His-tagged recombinant WT and different mutant variants of L13a proteins were expressed in Sf9 cells using the Bac-to-Bac baculoviral expression system following the manufacturer's protocol (Invitrogen). Briefly, Sf9 insect cells were transfected with WT or mutant L13a bacmids to generate baculoviral stocks. Sf9 cells were further infected with the baculoviral particles, lysed and His-tagged L13a proteins were purified using HisPur Ni-NTA purification Kit (Thermo Fisher Scientific). Purified proteins were tested using an in vitro translational silencing assay. Two hundred nanograms GAIT element bearing chimeric RNA transcript containing a GAIT element (Cap-Luc-GAIT-PolyA) and a cRNA transcript encoding T7 gene 10 (used as a loading and specificity control) were translated in rabbit reticulocyte lysate (RRL) in the presence of purified WT or mutant L13a proteins, 20 µM methionine-free amino acid mixture, 20 µCi translation grade S35 methionine, and 40U of RNasin in a total volume of 50 µL reaction at 30°C for 1 h. An aliquot was resolved by 10% SDS-PAGE. The gel was fixed, dried and radiolabeled bands were detected by autoradiography.
4
Purification of GST- and His-tagged Proteins
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Glutathione S-transferase (GST)- and polyhistidine (His)-tagged proteins were expressed in Rosetta 2(DE3) cells (EMD Millipore) growing in presence of chloramphenicol, ampicillin, and kanamycin (Sigma-Aldrich). Expression was induced by addition of 0.1 mM IPTG (AppliChem) for 6 h at 25°C or 18°C overnight. For purification of GST-tagged proteins, bacteria were suspended in Tris-buffered saline buffer containing PMSF and protease inhibitors (Roche) and subsequently sonificated. The supernatant was incubated with glutathione sepharose 4B (GE Healthcare) on an orbital mixer for 1 h at 4°C. Sepharose was washed and stored at 4°C until used. His-tagged proteins were purified using a His-Pur Ni-NTA Purification kit (Thermo Fisher Scientific) according to the manufacturer’s instruction. Expression, purity, and concentration were determined by SDS-PAGE using Coomassie Brilliant blue staining.
5
Transaminase Purification from Recombinant E. coli
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Cells of recombinant E. coli strain TMB2100 were harvested by centrifugation (3200 × g, 10 min) and washed once with 25 mL water. The cell pellet was resuspended in equilibration buffer, according to the manufacturer’s instructions, and subjected to sonication in a sonicator (Bandelin electronics, Berlin, Germany). Cell debris was removed by centrifugation (3200 × g, 10 min). The clear lysate supernatant was collected and passed through a nickel column (HisPur™ Ni-NTA Purification Kit, Thermo Scientific, Rockford, IL, USA), according to the description of the manufacturer. The purified transaminase was collected after elution with buffer, according to the manufacturer’s instructions. Protein concentration was determined using the Bradford method with bovine serum albumin (BSA) as a standard
[31 (link)]. Purity of the protein in the eluent was estimated with SDS-PAGE (Figure
2 ). The identity of the protein was confirmed by mass spectrometry at the SWEGENE proteomics platform (Lund, Sweden). The concentration of the purified transaminase was approximately 0.5 mg/ml.
[31 (link)]. Purity of the protein in the eluent was estimated with SDS-PAGE (Figure
6
In Vivo Protein-Protein Interaction Assay
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293T cells were transfected separately with GFP-tagged and 6xHis-tagged proteins. GFP-193a.a. and GFP were purified using anti-GFP antibody (#ab290, Abcam) and Catch and Release® v2.0 Reversible Immunoprecipitation System (Millipore, Burlington, MA, USA); 6xHis-SMO were purified with HisPur™ Ni-NTA Purification Kit (Thermo Fisher Scientific, Waltham, MA, USA). For in vivo binding assay, purified GFP or GFP-193a.a. were incubated with 6xHis-SMO for 4 h at 4 °C and then subjected to immunoprecipitation with the indicated primary antibodies at 4 °C overnight. Then, the protein complexes were collected by incubation with 30 μL protein A/G agarose (Gibco BRL, Grand Island, NY, USA) for 2 h at room temperature, followed by washing with cold PBST buffer 5 times and then subjected to western blotting.
7
Purification of His-tagged Adipsin Proteins
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HEK293 LTV cells were transfected with a pcDNA3.1/V5-HisB plasmid encoding either human or mouse cDNA using Lipofectamine 3000 transfection kit (L3000-015, Invitrogen, Carlsbad, CA, USA). The supernatant media was collected 48 h after transfection and cleared by two successive centrifugation, 2000× g at 4 °C for 10 min and 8500× g at 4 °C for 10 min. The His-tagged adipsin proteins were purified using a HisPur Ni-NTA purification kit (Thermo Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. Briefly, the HisPur Ni-NTA spin column was equilibrated with an equilibration buffer containing 10 mM imidazole. Approximately 16 mL of clarified medium was applied to a HisPur Ni-NTA spin column and incubated for 30 min at room temperature on a rotator. The medium was discarded by centrifugation at 700× g for 2 min. After three times washing using 25 mM imidazole containing wash buffer, the His-tagged protein was eluted using a 3 mL elution buffer containing 250 mM imidazole. In order to exchange the elution buffer and concentrate the product, the eluted fraction was applied to Amicon Ultra-4 centrifugal filter device with 10,000 molecular weight cutoff (MWCO) (No. UFC801008, Merck Millipore, Burlington, MA, USA) according to the manufacturer’s instructions.
8
Cloning and Purification of Key mTORC1/2 Proteins
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Mouse gene coding sequences for Sesn3, Akt1, Akt2, Sin1, Pras40, Protor1, and Pdpk1 were cloned by PCR using gene-specific primers (Supplementary Table 1 ) and inserted into a pcDNA3 mammalian expression vector that has an N-terminal FLAG or 3× HA tag. For imaging analysis, mouse Sesn3 was subcloned into a pLP-mCherry vector by PCR, and human RICTOR coding sequence (plasmid 11367; Addgene, Inc.) (37 (link)) was subcloned into a pEGFP vector using SalI and XmaI restriction enzyme sites. Mouse Sin1 and Protor1 coding sequences were also subcloned into a pEGFP vector by PCR. For recombinant protein preparation, mouse Sesn3, Sin1, Protor1, Rictor (COOH-terminal, 900 amino acids), and Akt1 coding sequences were subcloned into a pET-24 bacterial expression vector by PCR. To purify the recombinant proteins, BL21 bacteria were transformed with those plasmids and positive clones were selected for recombinant protein expression, with an overnight induction by isopropyl β-D-1-thiogalactopyranoside at 16°C. Bacteria were pelleted and homogenized in a lysis buffer (50 mmol/L NaH2PO4 [pH 7.4], 300 mmol/L NaCl, 10 mmol/L imidazole, 1 mmol/L phenylmethylsulfonyl fluoride), and the lysate was sonicated on ice. Since the recombinant protein has a 6× His tag, protein purification was performed using a HisPur Ni-NTA Purification Kit (Thermo Scientific).
9
Recombinant Protein Expression and Purification
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The construction of the pQE30 vector encoding rN, the transformation of Eschericia coli JM109 and the expression of rN were all done as previously described [41 (link)]. The rN protein was purified using the HisPur™ Ni-NTA Purification Kit according to the manufacturer’s instructions (Thermo Scientific™, Rockford, USA). The protein concentration of the eluted fractions was determined using the BCA™ protein assay kit (Thermo Scientific™) and the eluted fractions were analysed by 4–12% SDS-PAGE. The protein was visualised using colloid staining.
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