SbMYB44 open-reading frame (ORF) was PCR amplified using SbMYB44 PF (5′-CCGGAATTC ATGGAAAGCCACCAAACTTTA-3′) and PR (5′-CCGCTCGAG TTAATGGAAATGACCGTACCG-3′) with flanking restriction sites of EcoRI and XhoI sites, respectively, and cloned in pJET1.2 vector. The digested SbMYB44 was cloned in pET28a vector. Plasmids of pET28a-SbMYB44 and vector alone were transformed separately in Escherichia coli BL-21 Star (DE3) cells (Invitrogen). Recombinant protein was induced with 1 mM isopropyl β-d -1-thiogalactopyranoside (IPTG) at 37 °C for 2, 4, 6 h and purified under native conditions using Ni–NTA Fast Start Kit, using manufacturer's protocol (Qiagen, Germany).
Escherichia coli bl21 star de3 cells
Manufactured by Thermo Fisher Scientific
Sourced in United States
Escherichia coli BL21 Star (DE3) cells are a laboratory strain of E. coli bacteria commonly used for protein expression. They are designed to enhance the expression of recombinant proteins under the control of the T7 promoter system.
Automatically generated - may contain errors
Lab products found in correlation
Edta free protease inhibitor tablet, by Roche (3 mentions)
Emulsiflex c3, by Avestin (3 mentions)
Ni nta agarose bead, by Qiagen (2 mentions)
Ni nta fast start kit, by Qiagen (1 mentions)
Nhs biotin, by Thermo Fisher Scientific (1 mentions)
15n nh4cl, by Cambridge Isotopes (1 mentions)
13c glucose, by Cambridge Isotopes (1 mentions)
Superdex 75, by GE Healthcare (1 mentions)
Pet22b vector, by GenScript (1 mentions)
Cello oligosaccharides, by Megazyme (1 mentions)
Lightrun sequencing service, by Thermo Fisher Scientific (1 mentions)
Kta pure, by GE Healthcare (1 mentions)
Trans blot turbo mini pvdf, by Bio-Rad (1 mentions)
Horseradish peroxidase hrp linked secondary iggs, by Cell Signaling Technology (1 mentions)
Hiprep 26 60 sephacryl 100 hr column, by GE Healthcare (1 mentions)
Mini protean tgx stain free protein gel, by Bio-Rad (1 mentions)
Methyl 14c choline, by PerkinElmer (1 mentions)
Pet28a vector, by Thermo Fisher Scientific (1 mentions)
Rpmi 1640, by Biowest (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
14 protocols using escherichia coli bl21 star de3 cells
1
Cloning and Expression of SbMYB44 Protein
2
Biotin Labeling of Recombinant GAL-4
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Recombinant intact human GAL-4 (amino acids 1–323; NP_006140.1) without added tags was produced from vector pET28c in Escherichia coli BL21 Star (DE3) cells (Invitrogen) and then purified by affinity chromatography on lactosyl-sepharose as previously described for galectin-3 (70 (link)). A 20-fold molar excess of NHS-Biotin (Thermo Fisher Scientific) dissolved in dimethyl sulfoxide was added to a 2 mg/ml of GAL-4 solution in PBS buffer. After 2 h incubation on ice, labeled Gal-4 was separated from the unreacted dye by a buffer change to PBS on a PD10 column.
3
Purification of FAAP20 and Ubiquitin
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The His10-GB1-fused FAAP20 construct and the His6-tagged human ubiquitin were overexpressed in Escherichia coli BL21 STAR (DE3) cells (Invitrogen). Bacterial cells were cultured in M9 minimal media using 15N-NH4Cl and 13C-glucose as the sole nitrogen and carbon sources (Cambridge Isotope Laboratories), and induced by IPTG (0.1 mM IPTG at 20°C for 18 h for His10-GB1-fused FAAP20 and 1 mM IPTG at 20°C for 18 h for human ubiquitin). A total of 50 μM ZnSO4 was added to the cell culture at the time of induction for overexpression of His10-GB1-fused FAAP20. The overexpressed proteins were purified by a Ni2+-NTA column; then the N-terminal His6-tag of human ubiquitin and the N-terminal His10-GB1 tag of the FAAP20 construct were removed by thrombin and TEV cleavage, respectively. A benzamidine column was used to remove thrombin, and a second Ni2+-NTA column was used to remove protein tags (His6-tag from ubiquitin and His10-GB1-tag from FAAP20) and the TEV protease. Both proteins were further purified by size-exclusion chromatography (Superdex 75, GE Healthcare). Nuclear magnetic resonance (NMR) samples of the apo FAAP20 UBZ and the FAAP20 UBZ–ubiquitin complex were exchanged into an NMR buffer containing 25 mM sodium phosphate, 100 mM KCl and 10% D2O or 100% D2O (pH 7.0) and concentrated to final protein concentrations of 0.8–3 mM.
4
Purification of Histidine-Tagged FMRP Domains
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Escherichia coli BL21 star (DE3) cells (Invitrogen) were transformed with histidine-tagged pET151/D-Topo FMRP full-length, KH1, KH2 or RGG box constructs (6 (link)). The cells were grown in LB medium with ampicillin (100 mg/ml) and induced for 4 h at 20°C by addition of 1mM isopropyl-β-D-thiogalacto-pyranoside when the cultures reached an OD of 0.4 at 600 nm. Cells were then harvested and resuspended in lysis buffer [25 mM Tris–HCl pH 7.6, 300 mM KCl or LiCl, 1 mM EDTA, 1 mM dithiothreitol (DTT) , 20% glycerol, 5% NP-40, 0.5M urea, complete protease inhibitor cocktail (Roche), 1 mM PMSF] sonicated for 5 min and centrifuged at 15 000 r.p.m. for 30 min at 4°C. The supernatant was incubated with Ni-NTA Agarose beads (Qiagen) for 2 h at 4°C by agitation. Beads were washed four times at 4°C with washing buffer (25 mM Tris–HCl, pH 7.6, 300 mM KCl, 1 mM DTT, 0.5M urea, 20% glycerol, 20 mM imidazole). Fusion proteins were eluted from the beads with elution buffer (25 mM Tris–HCl pH 7.6, 50 mM KCl, 1 mM EDTA, 1 mM DTT, 10% glycerol, 0.1% Triton, 0.5M urea, 250 mM imidazole) for 30 min on ice.
5
Escherichia coli Protein Production
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A total of twelve monomeric variants isolated from the screening were subcloned from the pSCZ1 display vector into pET-26b( +). Soluble protein production in Escherichia coli BL21 Star (DE3) cells (Invitrogen) and purification by immobilized metal ion chromatography (IMAC) was performed as previously described13 (link).
6
Recombinant SARS-CoV-2 Nucleocapsid Protein Production
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
MBP‐tagged (pTHMT) full‐length and domain deletion SARS‐CoV‐2 nucleocapsid proteins were expressed in Escherichia coli BL21 Star (DE3) cells (Life Technologies). Bacterial cultures were grown to an optical density of 0.7–0.9 before induction with 1 mM isopropyl‐β‐d ‐1‐thiogalactopyranoside (IPTG) for 4 h at 37°C. Cell pellets were harvested by centrifugation and stored at −80°C. Cell pellets were resuspended in approximately 20 ml of 20 mM Tris 1M NaCl 10 mM imidazole pH 8.0 with one EDTA‐free protease inhibitor tablet (Roche) for approximately 2 g cell pellet and lysed using an Emulsiflex C3 (Avestin). The lysate was cleared by centrifugation at 47,850 g for 50 min at 4°C, filtered using a 0.2 μm syringe filter, and loaded onto a HisTrap HP 5 ml column. The protein was eluted with a gradient from 10 to 300 mM imidazole in 20 mM Tris 1.0 M NaCl pH 8.0. Fractions containing MBP‐N full‐length or domain deletions were loaded onto a HiLoad 26/600 Superdex 200 pg column equilibrated in 20 mM Tris, 1.0 M NaCl, 1 mM EDTA (added to further reduce protease degradation during processing), pH 8.0. Fractions with high purity were identified by SDS–PAGE and concentrated using a centrifugation filter with a 10 kDa cutoff (Amicon, Millipore).
7
Purification of CLAMP Transcription Factor
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
MBP-tagged CLAMP DBD was expressed and purified as described previously (Kaye et al., 2018 (link)). MBP-tagged (pTHMT, Peti and Page, 2007 (link)) FL CLAMP protein was expressed in Escherichia coli BL21 Star (DE3) cells (Life Technologies). Bacterial cultures were grown to an optical density of 0.7–0.9 before induction with 1 mM isopropyl-β-D-1-thiogalactopyranoside (IPTG) for 4 hr at 37°C.
Cell pellets were harvested by centrifugation and stored at −80°C. Cell pellets were resuspended in 20 mM Tris, 1 M NaCl, 0.1 mM ZnCl2, and 10 mM imidazole pH 8.0 with one EDTA-free protease inhibitor tablet (Roche) and lysed using an Emulsiflex C3 (Avestin). The lysate was cleared by centrifugation at 20,000 rpm for 50 min at 4°C, filtered using a 0.2 μm syringe filter, and loaded onto a HisTrap HP 5 ml column. The protein was eluted with a gradient from 10 to 300 mM imidazole in 20 mM Tris, 1.0 M NaCl pH 8.0, and 0.1 mM ZnCl2. Fractions containing MBP-CLAMP FL were loaded onto a HiLoad 26/600 Superdex 200 pg column equilibrated in 20 mM Tris, 1.0 M NaCl, pH 8.0. Fractions containing FL CLAMP were identified by SDS-PAGE and concentrated using a centrifugation filter with a 10-kDa cutoff (Amicon, Millipore) and frozen as aliquots.
Cell pellets were harvested by centrifugation and stored at −80°C. Cell pellets were resuspended in 20 mM Tris, 1 M NaCl, 0.1 mM ZnCl2, and 10 mM imidazole pH 8.0 with one EDTA-free protease inhibitor tablet (Roche) and lysed using an Emulsiflex C3 (Avestin). The lysate was cleared by centrifugation at 20,000 rpm for 50 min at 4°C, filtered using a 0.2 μm syringe filter, and loaded onto a HisTrap HP 5 ml column. The protein was eluted with a gradient from 10 to 300 mM imidazole in 20 mM Tris, 1.0 M NaCl pH 8.0, and 0.1 mM ZnCl2. Fractions containing MBP-CLAMP FL were loaded onto a HiLoad 26/600 Superdex 200 pg column equilibrated in 20 mM Tris, 1.0 M NaCl, pH 8.0. Fractions containing FL CLAMP were identified by SDS-PAGE and concentrated using a centrifugation filter with a 10-kDa cutoff (Amicon, Millipore) and frozen as aliquots.
8
Purification of SARS-CoV-2 Nucleocapsid Protein
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
MBP-tagged (pTHMT) full-length SARS-CoV2 nucleocapsid protein was expressed in Escherichia coli BL21 Star (DE3) cells (Life Technologies). Bacterial cultures were grown to an optical density of 0.7 to 0.9 before induction with 1 mM isopropyl-β-D-1-thiogalactopyranoside (IPTG) for 4 hrs at 37°C. Cell pellets were harvested by centrifugation and stored at −80°C. Cell pellets were resuspended in 20 mM Tris 1M NaCl 10 mM imidazole pH 8.0 with one EDTA-free protease inhibitor tablet (Roche) and lysed using an Emulsiflex C3 (Avestin). The lysate was cleared by centrifugation at 20,000 rpm for 50 min at 4°C, filtered using a 0.2 μm syringe filter, and loaded onto a HisTrap HP 5 mL column. The protein was eluted with a gradient from 10 to 300 mM imidazole in 20 mM Tris 1.0 M NaCl pH 8.0. Fractions containing MBP-N full-length were loaded onto a HiLoad 26/600 Superdex 200 pg column equilibrated in 20 mM Tris 1.0 M NaCl pH 8.0. Fractions with high purity were identified by SDS-PAGE and concentrated using a centrifugation filter with a 10 kDa cutoff (Amicon, Millipore).
9
Codon-optimized TfSOD Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The gene encoding TfSOD (Uniprot ID: Q47RC2) was codon optimized and cloned into the pET-22b(+) vector by Genscript (Piscataway, NJ, USA). Received plasmids were transformed into Escherichia coli BL21Star (DE3) cells (Life Technologies, Carlsbad, CA, USA).
10
Characterization of Cellulase Enzymes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Chemicals. Cello-oligosaccharides were obtained from Megazyme (Wicklow, Ireland).
All other chemicals were of analytical grade and purchased from standard manufacturers.
Enzymes. Initial hydrolysis of cellotetraose (Glc)4, cellopentaose (Glc)5, and (Glc)6 was conducted using TfCel5A-WT, TfCel9A-WT, and TfCel48A-WT. In the isothermal titration calorimetry (ITC) experiments, variants where the catalytic acid is mutated to alanine, a mutation that inactivates the enzyme, were used.
Cloning. Three gene constructs were ordered from Genscript; TfCel5A-WT (Uniprot ID: Q47RH8), TfCel9A-WT (Uniprot ID: Q47MW0), and TfCel48A-E359A. For TfCel48A-E359A, the glutamic acid residue in position 359 in the wild type gene (Uniprot ID: Q47NH7) was exchanged with an alanine. The signal peptides were removed from the genes encoding the enzymes before the genes were codon optimized and cloned into the pET-22b(+)-vector by GenScript (Piscataway, NJ, USA). The received plasmids were transformed into Escherichia coli BL21Star (DE3) cells as described by the manufacturer (Life Technologies, Carlsbad, CA, USA).
All other chemicals were of analytical grade and purchased from standard manufacturers.
Enzymes. Initial hydrolysis of cellotetraose (Glc)4, cellopentaose (Glc)5, and (Glc)6 was conducted using TfCel5A-WT, TfCel9A-WT, and TfCel48A-WT. In the isothermal titration calorimetry (ITC) experiments, variants where the catalytic acid is mutated to alanine, a mutation that inactivates the enzyme, were used.
Cloning. Three gene constructs were ordered from Genscript; TfCel5A-WT (Uniprot ID: Q47RH8), TfCel9A-WT (Uniprot ID: Q47MW0), and TfCel48A-E359A. For TfCel48A-E359A, the glutamic acid residue in position 359 in the wild type gene (Uniprot ID: Q47NH7) was exchanged with an alanine. The signal peptides were removed from the genes encoding the enzymes before the genes were codon optimized and cloned into the pET-22b(+)-vector by GenScript (Piscataway, NJ, USA). The received plasmids were transformed into Escherichia coli BL21Star (DE3) cells as described by the manufacturer (Life Technologies, Carlsbad, CA, USA).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!