The sequence of the mature PbMCS without mitochondrial import peptide was used for production of the recombinant enzyme in Escherichia coli. The coding sequence was amplified from cDNA of P. lutzii isolate Pb01 and cloned into a modified pET43 plasmid in frame with a His-Tag sequence (Hortschansky et al. 2007 (link)). The plasmid was transferred into E. coli BL21 (DE3) Rosetta 2 cells (Novagen/Merck), which were used for overproduction of the recombinant enzyme. Protein production was induced by cultivation in Overnight Express Instant TB medium at 30 °C. Cells were collected by centrifugation and resuspended in buffer A (50 mM Tris/HCl, 150 mM NaCl, 10% glycerol, pH 8.0), and disrupted by sonication. Lysates were clarified by centrifugation and loaded onto a nickel-Sepharose 6 Fast Flow gravity-flow column (GE Healthcare). After six washes with buffer A containing 30 mM imidazole the protein was eluted in buffer A containing 200 mM imidazole. The recombinant protein purity was assessed by SDS-PAGE. Fractions with purified enzyme were combined and desalted using centrifugal filter devices (Merck). Then, glycerol was added to purified MCS and samples were stored at − 20 °C. Protein concentrations were determined by using the Bradford protein assay kit from Bio-Rad and bovine serum albumin was employed as standard.
Centrifugal filter device
Manufactured by Merck Group
Sourced in United States, Germany
Centrifugal filter devices are laboratory equipment used to separate and concentrate molecules, particles, or cells from a solution or suspension. The core function of these devices is to apply centrifugal force to the sample, causing the denser components to move towards the bottom of the device, while the lighter components remain in the supernatant. This separation process allows for the isolation and purification of target analytes.
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Lab products found in correlation
Ni nta fast start kit, by Qiagen (2 mentions)
Hiprep 26 10 desalting column ge, by GE Healthcare (2 mentions)
Hispur ni nta resin, by Thermo Fisher Scientific (2 mentions)
Pet101 d topo vector, by Thermo Fisher Scientific (2 mentions)
Millex hv, by Merck Group (2 mentions)
Vcx130, by Sonics (2 mentions)
Bradford protein assay kit, by Bio-Rad (1 mentions)
E coli bl21 de3 rosetta 2 cells, by Merck Group (1 mentions)
Bl21 de3, by Tiangen Biotech (1 mentions)
Desalting column, by GE Healthcare (1 mentions)
Protein assay kit, by Bio-Rad (1 mentions)
Mascot search engine, by Matrix Science (1 mentions)
Protease inhibitor cocktail, by Avantor (1 mentions)
Gst resin, by GE Healthcare (1 mentions)
Glutathione sepharose, by Cytiva (1 mentions)
Sp sepharose fast flow, by GE Healthcare (1 mentions)
Nvt 90 rotor, by Beckman Coulter (1 mentions)
Phelper plasmid, by Agilent Technologies (1 mentions)
Pet28b, by GenScript (1 mentions)
Stainsall stain solution, by Merck Group (1 mentions)
50 protocols using centrifugal filter device
1
Recombinant PbMCS Enzyme Production
2
Overexpression and Purification of Recombinant Protein
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E. coli strain BL21 (DE3) obtained from Tiangen Biotech Co., Ltd (Beijing, China) was transformed with the plasmid and cultured in selective antibiotic LB agar plate. After 16 h, a single colony was picked and cultured in 10 mL LB medium containing 50 μg/mL kanamycin with vigorous shaking at 37 °C for 10 h. Then the 10 mL cultures were added to 250 mL media and cultured for 2 h. Next, protein expression was induced by adding IPTG to a final concentration of 0.5 mM. The cells were left to grow overnight at 16 °C and then harvested by centrifugation. The extraction and purification of protein were performed using Ni-NTA Fast Start Kit (QIAGEN, Duesseldorf, Germany). Then the purified protein was concentrated by Centrifugal Filter Devices (Merck Millipore, Billerica, MA, USA) and mixed with glycerol to a final concentration of 20%, and stored at −80 °C until used.
3
Purification of PsPIWI-RE Proteins
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For PsPIWI-RE protein expression, the gene encoding the full-length sequence for these proteins from P. stutzeri was optimised (sequences shown in Additional file 1 : Table S4) for E. coli codon usage and inserted into the plasmids pET-28a (+). PsPIWI-RE single point mutants (D525A, D610A, R639A, E718A, respectively) were synthesized by GenScript (Nanjing, China). The plasmid was transformed into E. coli BL21 (DE3) cells (Novagen). These cells were grown in 1 l Miller LB medium supplemented with kanamycin (50 μg/mL) at 37 °C. To induce expression of PsPIWI-RE, 0.5 mM isopropyl-β-d -thiogalactoside (IPTG) was added. After centrifugation, the collected cells were resuspended in 20 mM Tris–HCl (pH 8.0), 500 mM NaCl, 5 mM MgCl2, 2 mM 2-mercaptoethanol, and 10 mM imidazole (lysis buffer) and then lysed by high-pressure homogenisation. Protein purification was carried out using nickel-affinity chromatography, and then desalting was performed using a desalting column (GE Healthcare, USA). The purified protein was diluted to a final concentration of 1.5 mg mL–1 in 20 mM Tris–HCl pH 8.0, 500 mM NaCl, 5 mM MgCl2, and 2 mM DTT using centrifugal filter devices (Merck KGaA, Germany), and then aliquoted and stored at − 80 °C.
4
Extraction and Characterization of A. cantonensis L5 ESPs
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A. cantonensis L5 was collected from the host brains of infected rats. Next, the worms were incubated in fetal bovine serum (FBS)-free RPMI after washing with saline, phosphate-buffered saline, ddH2O, and RPMI. A. cantonensis L5 ESPs were concentrated by centrifugal filter devices (Merck Millipore, Darmstadt, Germany). The ESP concentration was determined by a Bio-Rad Protein Assay Kit (Bio-Rad, Hercules, CA, USA), according to the instructions of the manufacturer. Finally, it was employed to treat astrocytes and then observe cell survival and gene and protein expression.
5
Proteomic Analysis of DFSC Secretome
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Normal and inflamed DFSCs were cultured for 24 h, and the culture supernatants were collected. The proteins present in the cell culture supernatants were purified and concentrated using centrifugal filter devices (Merck, Billerica, MA, USA). Aliquots (25 μg) of the concentrated proteins were analysed and compared using 12% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by staining with Coomassie brilliant blue (CBB). Selected bands from 1-dimensional gel electrophoresis (1-DE) were digested with trypsin and analysed by LC-MS/MS. All tandem mass spectra were searched against the NCBI database using the Mascot search engine (Matrix Science, London, U.K.). Peptides with Mascot scores of 30 or above were identified.
6
Recombinant Protein Expression and Purification
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The empty and PMI-inserted pGEM-4 T-1 vectors were transformed into the E. coli strain Rosetta (DE3) plysS to express recombinant proteins. A 5-mL overnight liquid culture was used to inoculate 500 mL of lysogeny broth (LB) in 1-L Erlenmeyer flasks, and the cultures were grown at 37 °C until OD600 = 0.6 ~ 0.8. Then, induction with isopropyl-β-D-1-thiogalactopyranoside (IPTG) was performed at a final concentration of 1 mM. The cultures were then grown for an additional 20 h at 18 °C before harvesting. The cell pellet was suspended, washed and re-suspended with 10 mL of PBS buffer (pH 7.4) containing 1 mM phenylmethyl sulfonyl fluoride (PMSF), 1 mg/mL lysozyme and 1% protease inhibitor cocktails (AMRESCO, USA). Cell disruption was achieved by sonic vibration in an ultrasonic processor and centrifuged at 12000 rpm for 15 min to remove debris. The supernatants were then incubated with GST resin (GE Healthcare, USA) for 2 h, loaded onto a purification column and sequentially eluted with PBS buffer, low salt buffer (50 mM Tris-HCl, 10 mM reduced glutathione, pH 8.0) and high salt buffer (50 mM Tris-HCl, 20 mM reduced glutathione, 150 mM NaCl, pH 8.0) according to the standard protocol of the manufacturer. The recombinant proteins were desalted and concentrated by centrifugal filter devices (Millipore, USA).
7
Preparation of huMy9-6-IGN23 Conjugate
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Example 36
Preparation of huMy9-6-IGN23 Conjugate
A solution of huMy9-6 antibody at a concentration of 2 mg/mL in an aqueous buffer containing 0.05 M N-(2-hydroxyethyl)-piperazine-N′-2-ethanesulfonic acid (HEPES) and 2 mM ethylenediaminetetra-acetic acid (EDTA), pH 8.5 was treated with a 12.5-fold molar excess of a solution of IGN23-NHS in dimethylacetamide (DMA), glycerol, and sucrose. The final concentration of DMA, glycerol and sucrose in the buffer was 15%, 5% and 5% (v/v) respectively. The reaction mixture was stirred at room temperature for 120 min and then loaded onto a Sephadex G25 gel filtration column (HiPrep™ 26/10 Desalting Column GE #17-5087-01) that had been previously equilibrated into an aqueous buffer containing 10 mM histidine, 250 mM glycine, 1% sucrose, pH 5.5. The conjugated antibody-containing fractions were collected and pooled to yield product. The pooled sample was concentrated using Millipore centrifugal filter devices, and then dialyzed overnight against the same elution buffer to further purify the product.
The final conjugate was assayed spectrophotometrically using the extinction coefficients that were determined for IGN-23 (e330=15,231 M−1 cm−1 and e280=26,864 M−1 cm−1) and huMy9-6 (e280 nm=206,460 M−1cm−1). An average of 2.2 IGN23 molecules per molecule of antibody were linked.
8
Expressing and Purifying Recombinant Proteins
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The full-length coding sequences of PtrVCS2, PtrWOX13a, PtrZHD1, PtrGCN5-1 and PtrADA2b-3 were inserted into the pET101/D-TOPO vector (Invitrogen, K10101 ) for generating the 6×His tag fusion proteins. The primers used for construct generation are listed in Supplementary Table 6 . Recombinant proteins were expressed in the Escherichia coli BL21 strain, followed by purification with HisPur Ni-NTA Resin (Thermo Scientific, 88221). After washing and elution, the recombinant proteins were collected in PBS buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4) using Centrifugal Filter Devices (Millipore, UFC501096).
9
Zymography for MMP Activity Assessment
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In the present study, matrix metalloproteinase (MMP) activity in a conditioned medium was measured using zymography. Conditioned medium samples were collected and concentrated using centrifugal filter devices (Millipore, Bedford, MA, USA). Concentrated protein (50–60 μg) was loaded into NOVEX 12% zymogram casein gels (Invitrogen, Carlsbad, CA, USA). Proteins were electrophoresed in Tris/glycine sodium dodecyl sulfate (SDS) running buffer under nondenaturing conditions. Gels were washed thrice in 2.5% Triton X-100 for 30 min at room temperature to remove SDS. Zymograms were subsequently developed during incubation for 48 h at 37°C in zymogram reaction buffer [0.2 mol/L NaCl, 5 mmol/L CaCl2 , 40 mmol/L HCl, 50 mmol/L Tris, 0.02% Brij 35 (pH 7.1)]. Gels were stained with Coomassie Blue. Enzymatic activity was visualized as a clear band against a dark background of stained casein.
10
Purification of GST-Fused RKIP Mutant
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Recombinant glutathione-S-transferase (GST) and GST-fused mutant of Raf kinase inhibitory protein (RKIP-TV-delRBD) were purified from E. coli using glutathione sepharose (Amersham Biosciences). RKIP-TV-delRBD possesses a single mutation of threonine residue 153 to valine (Corbit et al., 2003 (link)) and a deletion mutation of amino acids 77–108, which include a main site for its association with Raf-1 (Yeung et al., 2000 (link)). Purified proteins were dialyzed in HEPES buffered solution (20 mM HEPES, pH 7.2), concentrated by centrifugal filter devices (Millipore), and stored at −80°C.
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