HT29, SKOV3, U87MG, A549, PC3, and MDA-MB-231 cell lines were purchased from the American Type Culture Collection (ATCC, Rockville, MD, USA) and cultured in different media with 10% FBS (HT29 and SKOV3 cells were cultured in McCoy’s 5A media, U87MG and A549 cells were cultured in MEM media, PC3 cells were cultured in F-12 media, and MDA-MB-231 cells were cultured in L-15 media. BL21 (DE3) competent cells were bought from Thermo Fisher Scientific.
Bl21 de3 competent cells
Manufactured by Thermo Fisher Scientific
Sourced in United States, Holy See (Vatican City State), United Kingdom
BL21 (DE3) competent cells are a bacterial strain commonly used for protein expression in molecular biology experiments. These cells are designed to efficiently express proteins under the control of the T7 promoter system. The BL21 (DE3) strain lacks the Lon and OmpT proteases, which can degrade expressed proteins, making it suitable for maintaining high levels of recombinant protein production.
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Lab products found in correlation
Bca kit, by Thermo Fisher Scientific (3 mentions)
Np osu, by Biosearch Technologies (2 mentions)
Mda mb 231, by American Type Culture Collection (1 mentions)
Skov3, by American Type Culture Collection (1 mentions)
U87mg, by American Type Culture Collection (1 mentions)
Ni nta agarose resin, by Qiagen (1 mentions)
Dithiothreitol (dtt), by Avantor (1 mentions)
Escherichia coli dh5α and, by Thermo Fisher Scientific (1 mentions)
Prism 7, by GraphPad (1 mentions)
Glutathione sepharose 4b, by GE Healthcare (1 mentions)
Isopropyl β d 1 thiogalactopyranoside (iptg), by Thermo Fisher Scientific (1 mentions)
Bl21 de3 plyss competent cells, by Thermo Fisher Scientific (1 mentions)
Sds page gel, by Bio-Rad (1 mentions)
U87mg human glioblastoma cell, by American Type Culture Collection (1 mentions)
Coomassie staining, by Bio-Rad (1 mentions)
Pet28b, by Agilent Technologies (1 mentions)
Quickchange 2 site directed mutagenesis kit, by Agilent Technologies (1 mentions)
Isopropyl d thiogalactopyranoside iptg, by Merck Group (1 mentions)
Geneart site directed mutagenesis system, by Thermo Fisher Scientific (1 mentions)
Pdest14, by Thermo Fisher Scientific (1 mentions)
16 protocols using bl21 de3 competent cells
1
Culturing Diverse Cell Lines in Optimal Media
2
Purification of GST- and His-tagged Proteins
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Plasmids expressing GST- and His- tagged proteins were transformed into BL21(DE3) competent cells (Thermo Scientific, EC0114) and grown in LB media at 37°C. Protein expression was induced by the addition of IPTG when the OD reached 0.6. After 3–4 h, cells were collected by centrifugation and resuspended in the appropriate lysis buffer. Cells were lysed using sonication and proteins were purified using standard protocols (46 (link),47 (link)).
3
Production and Characterization of LLO(LT) Conjugates
4
Production and Characterization of LLO(LT) Conjugates
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All peptides used in this study were purchased from Peptide 2.0 Inc., purified by reverse-phase high pressure liquid chromatography, and analyzed by mass-spectroscopy. A pET29b expression vector containing the His-tagged LLOLT sequence was provided by Dr. Emil Unanue (Washington University, St. Louis, MO)36 (link). For generation of LLOLT-N, site-directed mutagenesis was used to change the lysine at position 203 to an asparagine (N). Mutated clones were confirmed by sequencing. LLOLT and LLOLT-N were expressed in BL21 (DE3) competent cells (ThermoFisher) and purified as previously described36 (link). Protein purity was confirmed by SDS-PAGE. For conjugation of NP to LLOLT and LLOLT-N, 0.5mg of NP-OSu (LGC Biosearch Technologies) was added to 5mg of either LLOLT or LLOLT-N in 10 equal fractions over 20 minutes. The solution was then incubated at room temperature with rotations for 2 hours before being dialyzed into 0.1M NaHCO3, 145mM NaCl, pH 8.5. NP:LLOLT(-N) ratio was determined with the following extinction coefficients: LLOLT(-N) = 75750 M−1cm−1 and NP = 4230 M−1cm−1. Ratios of NP:LLOLT(-N) ranged from 5:1-10:1, and batches of NP-LLOLT(-N) were aliquoted and stored at -20°C to allow for continuity across experiments.
5
XptA2 Subcloning and Expression
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The full-length XptA2 wt DNA (WP_041979038.1.) was subcloned into the pET24a vector (Novagen) with a C-terminal 6× Histidine tag. The XptA2 2-fragment construct was synthesized by expressing the XptA2 wt sequence into two plasmids. The first 6039 base pairs were expressed in the kanamycin (Kan) selectable pET24a (Novagen) plasmid, and the last 549 base pairs were expressed in the ampicillin (Amp) selectable pET22b plasmid (Novagen) after adding an artificial start codon (methionine) at the beginning of the sequence. These plasmids were co-transformed into BL21(DE3) competent cells (Thermo Fisher Scientific, Waltham, MA, USA) and colonies were selected from plates containing both Amp and Kan to ensure that both plasmids were transformed into the cells.
6
Purification of GST- and His-tagged Proteins
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Plasmids expressing GST-and His-tagged proteins were transformed into BL21(DE3) competent cells (Thermo Scientific, EC0114) and grown in LB media at 37℃. Protein expression was induced by the addition of IPTG when the OD reached 0.6. After 3-4 hours, cells were collected by centrifugation and resuspended in the appropriate lysis buffer. Cells were lysed using sonication and proteins were purified using standard protocols (Harper and Speicher, 2011; Nallamsetty and Waugh, 2007) .
7
Engineered MEN1 Gene Constructs
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Plasmid design and construction was based on the original human MEN1 sequence first reported by Chandrasekharappa and colleagues and annotated in the GenBank database (U93236; ref. 21 (link)). This coding region of the human MEN1 gene (1,830 nucleotides) was ligated to the EcoRI site of the pcDNA3.1+/C-(K)-DYK plasmid upstream of the FLAG-epitope DYKDDDDK sequence. Two single-nucleotide mutations and a third single-nucleotide variant previously identified by WES of patients with GEP-NETs (20 ) were introduced as follows: c.1546dupC (R516fs), c.703G>A (E235K), and c.1621G>A (A541T). As the c.1546dupC mutation encodes a premature stop codon that results in protein truncation at amino acid 529, the FLAG-epitope sequence was introduced at the N-terminal domain. Plasmid construction and validation were performed by GenScript (Genscript Biotech Corp). BL21 (DE3) competent cells (Invitrogen) were transformed with the pcDNA empty vector or the vector expressing wild-type MEN1 containing the respective mutations. Plasmids were purified from transformed Escherichia coli following ampicillin selection using the QIAGEN Plasmid Purification Midi-Prep Kit (QIAGEN) and reconstituted in sterile Tris-EDTA buffer.
8
Purification and Characterization of MoaA
9
Recombinant Protein Expression and Purification
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The plasmids encoding GST, Pen (FL)-GST, Pen (1-63)-GST, Pen (1-275)-GST, Pen (1-447)-GST, Pen (234-552)-GST, human Kpna2-GST, MBP, and MBP-Karyβ3 were transformed into BL21(DE3)-competent cells (Invitrogen) for protein expression. Fusion protein expression was induced by treatment with 1 mM IPTG (isopropyl-β-d -thiogalactopyranoside) for 24 hours at 18°C. After induction, Escherichia coli were resuspended in ice-cold lysis buffer [1× PBS (pH 7.4), 0.3% Triton, 150 mM NaCl, 10% glycerol, 10 mg/ml lysozyme, and 5 mM EDTA] and then sonicated using a ChromTech UP-500 system (10 cycles at 30% amplitude for 5 s on/5 s off). After sonication, lysates were centrifuged at 12,000g at 4°C for 20 min. Supernatants were subjected to affinity purification using Ni2+ resin (Invitrogen) or Glutathione Sepharose 4B (GE Healthcare) to collect recombinant proteins. Before pull-down assays, beads binding to recombinant proteins were stored in binding buffer (50 mM tris-Cl, 150 mM NaCl, 0.05% SDS, 1% Triton X-100, and 1× Roche EDTA-free protease inhibitor).
10
Recombinant protein expression in E. coli
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Bacterial expression plasmids encoding the six-Histidine tagged SodA (rSodA) (BEI Resources, Manassas, VA) were transformed into BL21/DE3, pLysS competent cells (Invitrogen Inc., Carlsbad, CA). Bacterial expression plasmids encoding the six-Histidine tagged KatG (rKatG) (BEI Resources, Manassas, VA) were transformed into BL21/DE3 competent cells (Invitrogen Inc., Carlsbad, CA). Once bacterial density reached an A600 of 0.4 to 0.6, 0.2 mM to 1 mM IPTG (ThermoFisher Scientific, Waltham, MA) was added to induce recombinant protein expression. The culture was harvested after overnight incubation at 37°C with shaking at 200 rpm.
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