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Promega e coli bl21
E Coli Bl21, supplied by Promega, used in various techniques. Bioz Stars score: 95/100, based on 21 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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e coli bl21 - by Bioz Stars, 2020-01
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Transduction:

Article Title: RsgA releases RbfA from 30S ribosome during a late stage of ribosome biosynthesis
Article Snippet: W3110Δ rsgA Δ rbfA was constructed by transferring the kanamycin-resistant marker of W3110Δ rsgA into W3110Δ rbfA via P1 transduction. .. For expression of recombinant RsgA and RbfA, we used E. coli BL21(DE3) (Promega Corporation).

Clone Assay:

Article Title: Identification and in vitro Analysis of the GatD/MurT Enzyme-Complex Catalyzing Lipid II Amidation in Staphylococcus aureusIdentification of Genetic Determinants and Enzymes Involved with the Amidation of Glutamic Acid Residues in the Peptidoglycan of Staphylococcus aureus
Article Snippet: Paragraph title: Cloning, overexpression and purification of S. aureus murT , gatD and glnA as His6-tag fusions ... E. coli BL21(DE3) (Promega) cells transformed with the appropriate recombinant plasmid were grown in LB-medium (50 µg/ml ampicillin) at 37°C.

Article Title: The pH Sensitivity of Murine Heat Shock Protein 47 (HSP47) Binding to Collagen Is Affected by Mutations in the Breach Histidine Cluster *
Article Snippet: Paragraph title: Cloning and Site-directed Mutagenesis ... HSP47 WT and HA mutants were expressed in E. coli BL21(DE3)pLysS (Promega, WI).

Article Title: The Dengue ED3 Dot Assay, a Novel Serological Test for the Detection of Denguevirus Type-Specific Antibodies and Its Application in a Retrospective Seroprevalence Study
Article Snippet: After BamHI and HindIII digestion, the PCR fragment was cloned into pMAL-p4x vector (New England Biolabs) downstream to the malE gene [ ]. .. For expression of the MBP-ED3-fusion proteins, pMAL-p4x plasmids containing the insert of choice were transformed into E. coli BL21(DE3) (Promega, Mannheim, Germany) cells.

Article Title: Recombinant N-acyl homoserine lactone-Lactonase AiiAQSI-1 Attenuates Aeromonas hydrophila Virulence Factors, Biofilm Formation and Reduces Mortality in Crucian Carp
Article Snippet: Paragraph title: 3.2. Cloning, Expression, and Purification of AiiAQSI-1 Protein ... The recombinant pET-30a-aiiAQSI-1 vector was then transformed into E. coli BL21(DE3) (Promega, Madison WI,, USA).

Article Title: Identification and in vitro Analysis of the GatD/MurT Enzyme-Complex Catalyzing Lipid II Amidation in Staphylococcus aureusIdentification of Genetic Determinants and Enzymes Involved with the Amidation of Glutamic Acid Residues in the Peptidoglycan of Staphylococcus aureus
Article Snippet: Co-purification of the GatD/MurT enzyme complex The gatD/murT operon was amplified using primers murT-for and gatD-rev ( ) and cloned into pET21b vector (Novagen) generating a C-terminal gatD -His tag fusion. .. The corresponding plasmid was introduced into E. coli BL21 (Promega) and overexpression was carried out as described above.

Article Title: Physical Interaction between Coat Morphogenetic Proteins SpoVID and CotE Is Necessary for Spore Encasement in Bacillus subtilis
Article Snippet: Escherichia coli DH5α (Invitrogen) was used for cloning. .. E. coli BL21(DE3) (Promega) was used for expression of cotE-STOP and spoVID-his constructs (see below).

Centrifugation:

Article Title: The Dengue ED3 Dot Assay, a Novel Serological Test for the Detection of Denguevirus Type-Specific Antibodies and Its Application in a Retrospective Seroprevalence Study
Article Snippet: For expression of the MBP-ED3-fusion proteins, pMAL-p4x plasmids containing the insert of choice were transformed into E. coli BL21(DE3) (Promega, Mannheim, Germany) cells. .. The bacteria were harvested after four hours via centrifugation at 10,000 rpm for 3 min (Beckman Coulter Avanti J-26 XP, rotor JA-14) and were suspended in lysis buffer (20 mM Tris pH 7.4, 200 mM NaCl, 1 mM EDTA, 5 mg/mL lysozyme).

Article Title: Functional and Biochemical Analysis of Chlamydia trachomatis MurC, an Enzyme Displaying UDP-N-Acetylmuramate:Amino Acid Ligase Activity
Article Snippet: For recombinant overexpression and purification of the C. trachomatis MurC domain, plasmid pCTHisSMurC was transformed into E. coli BL21(DE3) (Promega) and grown in 10 liters of Terrific broth ( ) at 37°C until an A 600 of 0.6 was reached. .. Cultures were induced with 1 mM IPTG and grown for a further 3 h before bacteria were harvested by centrifugation.

Amplification:

Article Title: A male sterility-associated cytotoxic protein ORF288 in Brassica juncea causes aborted pollen development
Article Snippet: Expression of orf288 in E. coli The whole fragment of orf288 was amplified from the cDNA of flower buds using the primer pairs KpnI288STR and BamHI288ED; in Supplementary Table S1 , available at JXB online, the Kpn I and Bam HI sites in the primer sequences are underlined. .. The expression of ORF288 in E. coli BL21(DE3)plysS (Promega) was induced by adding 0.5 mM isopropyl-β-D -thiogalactopyranoside (IPTG) when the optical density (OD) of the samples reached ∼0.6.

Article Title: Identification and in vitro Analysis of the GatD/MurT Enzyme-Complex Catalyzing Lipid II Amidation in Staphylococcus aureusIdentification of Genetic Determinants and Enzymes Involved with the Amidation of Glutamic Acid Residues in the Peptidoglycan of Staphylococcus aureus
Article Snippet: Cloning, overexpression and purification of S. aureus murT , gatD and glnA as His6-tag fusions S. aureus N315 murT (SA1708), gatD (SA1707) and glnA were amplified using forward and reverse primers as listed in and cloned into a pET21b vector (Novagen) using NdeI and XhoI restriction sites to generate C-terminal His6 -fusion proteins. .. E. coli BL21(DE3) (Promega) cells transformed with the appropriate recombinant plasmid were grown in LB-medium (50 µg/ml ampicillin) at 37°C.

Article Title: The pH Sensitivity of Murine Heat Shock Protein 47 (HSP47) Binding to Collagen Is Affected by Mutations in the Breach Histidine Cluster *
Article Snippet: The mouse HSP47 cDNA was amplified by PCR using 5′ and 3′ primers with HindIII and NdeI restriction sites, respectively. .. HSP47 WT and HA mutants were expressed in E. coli BL21(DE3)pLysS (Promega, WI).

Article Title: The Dengue ED3 Dot Assay, a Novel Serological Test for the Detection of Denguevirus Type-Specific Antibodies and Its Application in a Retrospective Seroprevalence Study
Article Snippet: The ED3 coding region was amplified from cDNA with specific primers and Phusion High-Fidelity DNA Polymerase (New England Biolabs, Frankfurt am Main, Germany). .. For expression of the MBP-ED3-fusion proteins, pMAL-p4x plasmids containing the insert of choice were transformed into E. coli BL21(DE3) (Promega, Mannheim, Germany) cells.

Article Title: Recombinant N-acyl homoserine lactone-Lactonase AiiAQSI-1 Attenuates Aeromonas hydrophila Virulence Factors, Biofilm Formation and Reduces Mortality in Crucian Carp
Article Snippet: Cloning, Expression, and Purification of AiiAQSI-1 Protein Total genomic DNA from Bacillus sp. strain QSI-1 was extracted using an Ezup Column Bacteria Genomic DNA Purification Kit (Sangon Biotech, Shanghai, China) and used as template for amplification of aiiA gene by PCR. .. The recombinant pET-30a-aiiAQSI-1 vector was then transformed into E. coli BL21(DE3) (Promega, Madison WI,, USA).

Article Title: Identification and in vitro Analysis of the GatD/MurT Enzyme-Complex Catalyzing Lipid II Amidation in Staphylococcus aureusIdentification of Genetic Determinants and Enzymes Involved with the Amidation of Glutamic Acid Residues in the Peptidoglycan of Staphylococcus aureus
Article Snippet: Co-purification of the GatD/MurT enzyme complex The gatD/murT operon was amplified using primers murT-for and gatD-rev ( ) and cloned into pET21b vector (Novagen) generating a C-terminal gatD -His tag fusion. .. The corresponding plasmid was introduced into E. coli BL21 (Promega) and overexpression was carried out as described above.

Construct:

Article Title: The pH Sensitivity of Murine Heat Shock Protein 47 (HSP47) Binding to Collagen Is Affected by Mutations in the Breach Histidine Cluster *
Article Snippet: Each construct was sequenced to confirm the presence of each mutation (and no others) between the promoter and terminator regions. .. HSP47 WT and HA mutants were expressed in E. coli BL21(DE3)pLysS (Promega, WI).

Article Title: The Dengue ED3 Dot Assay, a Novel Serological Test for the Detection of Denguevirus Type-Specific Antibodies and Its Application in a Retrospective Seroprevalence Study
Article Snippet: All MBP constructs were sequenced to verify the respective identity (LGC genomics, Berlin, Germany). .. For expression of the MBP-ED3-fusion proteins, pMAL-p4x plasmids containing the insert of choice were transformed into E. coli BL21(DE3) (Promega, Mannheim, Germany) cells.

Article Title: RsgA releases RbfA from 30S ribosome during a late stage of ribosome biosynthesis
Article Snippet: W3110Δ rsgA Δ rbfA was constructed by transferring the kanamycin-resistant marker of W3110Δ rsgA into W3110Δ rbfA via P1 transduction. .. For expression of recombinant RsgA and RbfA, we used E. coli BL21(DE3) (Promega Corporation).

Article Title: Physical Interaction between Coat Morphogenetic Proteins SpoVID and CotE Is Necessary for Spore Encasement in Bacillus subtilis
Article Snippet: .. E. coli BL21(DE3) (Promega) was used for expression of cotE-STOP and spoVID-his constructs (see below). ..

Incubation:

Article Title: Identification and in vitro Analysis of the GatD/MurT Enzyme-Complex Catalyzing Lipid II Amidation in Staphylococcus aureusIdentification of Genetic Determinants and Enzymes Involved with the Amidation of Glutamic Acid Residues in the Peptidoglycan of Staphylococcus aureus
Article Snippet: E. coli BL21(DE3) (Promega) cells transformed with the appropriate recombinant plasmid were grown in LB-medium (50 µg/ml ampicillin) at 37°C. .. Aliquots of 200 mg/ml lysozyme, 100 mg/ml DNase and 10 mg/ml RNase were added; cells were incubated for 30 min on ice and sonicated.

Article Title: Identification of a Cytoplasmic Complex That Adds a Cap onto 5?-Monophosphate RNA ▿Identification of a Cytoplasmic Complex That Adds a Cap onto 5?-Monophosphate RNA ▿ §
Article Snippet: Escherichia coli BL21(DE3)pLysS (Promega) was transformed with the plasmid pGEX-meIF4E, expressing a glutathione S -transferase (GST) fusion protein with murine eIF4E cap-binding protein (GST-eIF4E). .. Cleared bacterial lysate was incubated with m7 GTP-Sepharose (GE Biosciences) by end-over-end rotation at 4°C overnight.

Article Title: The Dengue ED3 Dot Assay, a Novel Serological Test for the Detection of Denguevirus Type-Specific Antibodies and Its Application in a Retrospective Seroprevalence Study
Article Snippet: For expression of the MBP-ED3-fusion proteins, pMAL-p4x plasmids containing the insert of choice were transformed into E. coli BL21(DE3) (Promega, Mannheim, Germany) cells. .. A volume of 2.7 L of dyt-medium supplemented with 300 µg/mL ampicillin, 0.5 mM IPTG and 0.2% glucose was inoculated 1:100 with an overnight culture and incubated at 37 °C in a horizontal shaker (GFL3031, GFL, Burgwedel, Germany) at 180 rpm.

Expressing:

Article Title: A male sterility-associated cytotoxic protein ORF288 in Brassica juncea causes aborted pollen development
Article Snippet: .. The expression of ORF288 in E. coli BL21(DE3)plysS (Promega) was induced by adding 0.5 mM isopropyl-β-D -thiogalactopyranoside (IPTG) when the optical density (OD) of the samples reached ∼0.6. .. Over the next 2.5 h, the OD of the samples was measured every 30 min at 600 nm using a UV-1601 spectrophotometer (Shimadzu, Japan)

Article Title: Identification and in vitro Analysis of the GatD/MurT Enzyme-Complex Catalyzing Lipid II Amidation in Staphylococcus aureusIdentification of Genetic Determinants and Enzymes Involved with the Amidation of Glutamic Acid Residues in the Peptidoglycan of Staphylococcus aureus
Article Snippet: E. coli BL21(DE3) (Promega) cells transformed with the appropriate recombinant plasmid were grown in LB-medium (50 µg/ml ampicillin) at 37°C. .. At an OD600 of 0.6, IPTG was added at a concentration of 0.75 mM to induce expression of the recombinant proteins.

Article Title: Identification of a Cytoplasmic Complex That Adds a Cap onto 5?-Monophosphate RNA ▿Identification of a Cytoplasmic Complex That Adds a Cap onto 5?-Monophosphate RNA ▿ §
Article Snippet: .. Escherichia coli BL21(DE3)pLysS (Promega) was transformed with the plasmid pGEX-meIF4E, expressing a glutathione S -transferase (GST) fusion protein with murine eIF4E cap-binding protein (GST-eIF4E). .. Cleared bacterial lysate was incubated with m7 GTP-Sepharose (GE Biosciences) by end-over-end rotation at 4°C overnight.

Article Title: The Dengue ED3 Dot Assay, a Novel Serological Test for the Detection of Denguevirus Type-Specific Antibodies and Its Application in a Retrospective Seroprevalence Study
Article Snippet: .. For expression of the MBP-ED3-fusion proteins, pMAL-p4x plasmids containing the insert of choice were transformed into E. coli BL21(DE3) (Promega, Mannheim, Germany) cells. .. A volume of 2.7 L of dyt-medium supplemented with 300 µg/mL ampicillin, 0.5 mM IPTG and 0.2% glucose was inoculated 1:100 with an overnight culture and incubated at 37 °C in a horizontal shaker (GFL3031, GFL, Burgwedel, Germany) at 180 rpm.

Article Title: Recombinant N-acyl homoserine lactone-Lactonase AiiAQSI-1 Attenuates Aeromonas hydrophila Virulence Factors, Biofilm Formation and Reduces Mortality in Crucian Carp
Article Snippet: Paragraph title: 3.2. Cloning, Expression, and Purification of AiiAQSI-1 Protein ... The recombinant pET-30a-aiiAQSI-1 vector was then transformed into E. coli BL21(DE3) (Promega, Madison WI,, USA).

Article Title: Rare human Caspase-6-R65W and Caspase-6-G66R variants identify a novel regulatory region of Caspase-6 activity
Article Snippet: .. Protein expression and purification Full-length Casp6 was expressed at 30 °C in E. coli BL21(DE3)pLysS (Promega, Fitchburg, WI, USA) and purified using Ni Sepharose Fast Flow 6 (GE Healthcare, Little Chalfont, UK) using Tris buffers pH 8.0. .. Eluted Casp6 was diluted fivefold with buffer A1 (20 mM Tris pH 8.5, 2 mM DTT, 5% glycerol), loaded on Macro Prep High Q resin (Bio-Rad Laboratories, Hercules, CA, USA), washed with 20 mM Tris pH 8.5, 2 mM DTT, 5% glycerol, 50 mM NaCl and eluted with 80-260 mM NaCl gradient in buffer A1.

Article Title: RsgA releases RbfA from 30S ribosome during a late stage of ribosome biosynthesis
Article Snippet: .. For expression of recombinant RsgA and RbfA, we used E. coli BL21(DE3) (Promega Corporation). .. Cultures of constructed strains were grown at 37°C in LB medium with agitation; ampicillin (50 μg/ml) was added to the medium for strains harbouring a plasmid with an ampicillin-resistant marker (pUC26-2, pMW118, pMWrsgA, pMWrbfA+ or its variants and pGEMrbfA or its variants), and chloramphenicol (10 μg/ml) was added to the medium for strains harbouring a plasmid with a chloramphenicol-resistant marker (pCA24N– and pCArbfA).

Article Title: Rare human Caspase-6-R65W and Caspase-6-G66R variants identify a novel regulatory region of Caspase-6 activity
Article Snippet: Paragraph title: Protein expression and purification ... Full-length Casp6 was expressed at 30 °C in E. coli BL21(DE3)pLysS (Promega, Fitchburg, WI, USA) and purified using Ni Sepharose Fast Flow 6 (GE Healthcare, Little Chalfont, UK) using Tris buffers pH 8.0.

Article Title: Flavonolignans As a Novel Class of Sodium Pump Inhibitors
Article Snippet: .. Expression and purification of the isolated large cytoplasmic segment C45 The large cytoplasmic segment connecting the transmembrane helices 4 and 5 (C45 loop, residues L354-I777 of the mouse brain sequence) with a (His)6 -tag at the N-terminus was expressed in E. coli BL21 (Promega, USA) and purified using a Co2+ -based affinity resin (Clontech, USA) as described previously (Grycova et al., ). ..

Article Title: Physical Interaction between Coat Morphogenetic Proteins SpoVID and CotE Is Necessary for Spore Encasement in Bacillus subtilis
Article Snippet: .. E. coli BL21(DE3) (Promega) was used for expression of cotE-STOP and spoVID-his constructs (see below). ..

Bradford Assay:

Article Title: Flavonolignans As a Novel Class of Sodium Pump Inhibitors
Article Snippet: Expression and purification of the isolated large cytoplasmic segment C45 The large cytoplasmic segment connecting the transmembrane helices 4 and 5 (C45 loop, residues L354-I777 of the mouse brain sequence) with a (His)6 -tag at the N-terminus was expressed in E. coli BL21 (Promega, USA) and purified using a Co2+ -based affinity resin (Clontech, USA) as described previously (Grycova et al., ). .. Protein were analyzed by Coomassie blue stained SDS PAGE and concentration was determined using the Bradford assay (Bradford, ) using BSA as a standard.

Transformation Assay:

Article Title: Identification and in vitro Analysis of the GatD/MurT Enzyme-Complex Catalyzing Lipid II Amidation in Staphylococcus aureusIdentification of Genetic Determinants and Enzymes Involved with the Amidation of Glutamic Acid Residues in the Peptidoglycan of Staphylococcus aureus
Article Snippet: .. E. coli BL21(DE3) (Promega) cells transformed with the appropriate recombinant plasmid were grown in LB-medium (50 µg/ml ampicillin) at 37°C. .. At an OD600 of 0.6, IPTG was added at a concentration of 0.75 mM to induce expression of the recombinant proteins.

Article Title: Identification of a Cytoplasmic Complex That Adds a Cap onto 5?-Monophosphate RNA ▿Identification of a Cytoplasmic Complex That Adds a Cap onto 5?-Monophosphate RNA ▿ §
Article Snippet: .. Escherichia coli BL21(DE3)pLysS (Promega) was transformed with the plasmid pGEX-meIF4E, expressing a glutathione S -transferase (GST) fusion protein with murine eIF4E cap-binding protein (GST-eIF4E). .. Cleared bacterial lysate was incubated with m7 GTP-Sepharose (GE Biosciences) by end-over-end rotation at 4°C overnight.

Article Title: The Dengue ED3 Dot Assay, a Novel Serological Test for the Detection of Denguevirus Type-Specific Antibodies and Its Application in a Retrospective Seroprevalence Study
Article Snippet: .. For expression of the MBP-ED3-fusion proteins, pMAL-p4x plasmids containing the insert of choice were transformed into E. coli BL21(DE3) (Promega, Mannheim, Germany) cells. .. A volume of 2.7 L of dyt-medium supplemented with 300 µg/mL ampicillin, 0.5 mM IPTG and 0.2% glucose was inoculated 1:100 with an overnight culture and incubated at 37 °C in a horizontal shaker (GFL3031, GFL, Burgwedel, Germany) at 180 rpm.

Article Title: Recombinant N-acyl homoserine lactone-Lactonase AiiAQSI-1 Attenuates Aeromonas hydrophila Virulence Factors, Biofilm Formation and Reduces Mortality in Crucian Carp
Article Snippet: .. The recombinant pET-30a-aiiAQSI-1 vector was then transformed into E. coli BL21(DE3) (Promega, Madison WI,, USA). .. After inducible expression with 0.5 mM IPTG at 37 °C for 4 h, cells were harvested and disrupted by sonication.

Article Title: Functional and Biochemical Analysis of Chlamydia trachomatis MurC, an Enzyme Displaying UDP-N-Acetylmuramate:Amino Acid Ligase Activity
Article Snippet: .. For recombinant overexpression and purification of the C. trachomatis MurC domain, plasmid pCTHisSMurC was transformed into E. coli BL21(DE3) (Promega) and grown in 10 liters of Terrific broth ( ) at 37°C until an A 600 of 0.6 was reached. .. Cultures were induced with 1 mM IPTG and grown for a further 3 h before bacteria were harvested by centrifugation.

Article Title: Glucose-Specific Enzyme IIA of the Phosphoenolpyruvate:Carbohydrate Phosphotransferase System Modulates Chitin Signaling Pathways in Vibrio cholerae
Article Snippet: MarC9 (a mutant protein with Himar1 transposase) N-terminally fused to the maltose-binding protein (MBP::MarC9) was purified from E. coli BL21(DE3) (Promega) carrying pMAL-SYMarC9 as described previously ( ). .. Mutagenized DNA was introduced into SY0638S by natural transformation, and then the bacterial culture was directly plated onto LA containing S-Gal (Sigma) and Cm to determine the Lac phenotypes of transformants.

Over Expression:

Article Title: Identification and in vitro Analysis of the GatD/MurT Enzyme-Complex Catalyzing Lipid II Amidation in Staphylococcus aureusIdentification of Genetic Determinants and Enzymes Involved with the Amidation of Glutamic Acid Residues in the Peptidoglycan of Staphylococcus aureus
Article Snippet: Paragraph title: Cloning, overexpression and purification of S. aureus murT , gatD and glnA as His6-tag fusions ... E. coli BL21(DE3) (Promega) cells transformed with the appropriate recombinant plasmid were grown in LB-medium (50 µg/ml ampicillin) at 37°C.

Article Title: Identification and in vitro Analysis of the GatD/MurT Enzyme-Complex Catalyzing Lipid II Amidation in Staphylococcus aureusIdentification of Genetic Determinants and Enzymes Involved with the Amidation of Glutamic Acid Residues in the Peptidoglycan of Staphylococcus aureus
Article Snippet: .. The corresponding plasmid was introduced into E. coli BL21 (Promega) and overexpression was carried out as described above. .. Co-elution from Ni-NTA column (Qiagen) of the GatD-His6 /MurT complex was analyzed by SDS page.

Article Title: Functional and Biochemical Analysis of Chlamydia trachomatis MurC, an Enzyme Displaying UDP-N-Acetylmuramate:Amino Acid Ligase Activity
Article Snippet: .. For recombinant overexpression and purification of the C. trachomatis MurC domain, plasmid pCTHisSMurC was transformed into E. coli BL21(DE3) (Promega) and grown in 10 liters of Terrific broth ( ) at 37°C until an A 600 of 0.6 was reached. .. Cultures were induced with 1 mM IPTG and grown for a further 3 h before bacteria were harvested by centrifugation.

Countercurrent Chromatography:

Article Title: The pH Sensitivity of Murine Heat Shock Protein 47 (HSP47) Binding to Collagen Is Affected by Mutations in the Breach Histidine Cluster *
Article Snippet: The primers used were as follows (with the introduced mutation sites underlined ): H191A, 5′-ATG TTC TTT AAG CCA GCA TGG GAT GAG AAG TTT; H197A, 5′-CA CAC TGG GAT GAG AAG TTT CAC GCA AAG ATG GTG G; H198A, 5′-CA CAC TGG GAT GAG AAG TTT GCA CAC AAG ATG GTG G; H197A,H198A, 5′-CA CAC TGG GAT GAG AAG TTT GCA GCA AAG ATG GTG G; H244A, 5′-G ATG CCC CTG GCT GCA AAG CTC TCC AGC; H255A,H256A, 5′-GC CTC ATC ATC CTC ATG CCC GCA GCA GTG GAG CCG C; H302A, 5′-CAT GAC CTG CAG AAA GCC CTG GCA GGA; W110F, 5′-ACT GCG CGC AAC GTG ACC TTT AAA CTG GGC AGC CGC; W158F, 5′-TCC ATC AAC GAG TTT GCC TCG CAG ACC ACG; W192F, 5′-TTC TTT AAG CCA CAC TTT GAT GAG AAG TTT CAG; W275F, 5′-GAG CAG CTG AAG GCC TTT ATG GGA AAG ATG CAG AAG; W341F, 5′-ACT GCC TTC GAG TTC GAC ACC GAG GGC AAC. .. HSP47 WT and HA mutants were expressed in E. coli BL21(DE3)pLysS (Promega, WI).

Flow Cytometry:

Article Title: Rare human Caspase-6-R65W and Caspase-6-G66R variants identify a novel regulatory region of Caspase-6 activity
Article Snippet: .. Protein expression and purification Full-length Casp6 was expressed at 30 °C in E. coli BL21(DE3)pLysS (Promega, Fitchburg, WI, USA) and purified using Ni Sepharose Fast Flow 6 (GE Healthcare, Little Chalfont, UK) using Tris buffers pH 8.0. .. Eluted Casp6 was diluted fivefold with buffer A1 (20 mM Tris pH 8.5, 2 mM DTT, 5% glycerol), loaded on Macro Prep High Q resin (Bio-Rad Laboratories, Hercules, CA, USA), washed with 20 mM Tris pH 8.5, 2 mM DTT, 5% glycerol, 50 mM NaCl and eluted with 80-260 mM NaCl gradient in buffer A1.

Article Title: Rare human Caspase-6-R65W and Caspase-6-G66R variants identify a novel regulatory region of Caspase-6 activity
Article Snippet: .. Full-length Casp6 was expressed at 30 °C in E. coli BL21(DE3)pLysS (Promega, Fitchburg, WI, USA) and purified using Ni Sepharose Fast Flow 6 (GE Healthcare, Little Chalfont, UK) using Tris buffers pH 8.0. .. Eluted Casp6 was diluted fivefold with buffer A1 (20 mM Tris pH 8.5, 2 mM DTT, 5% glycerol), loaded on Macro Prep High Q resin (Bio-Rad Laboratories, Hercules, CA, USA), washed with 20 mM Tris pH 8.5, 2 mM DTT, 5% glycerol, 50 mM NaCl and eluted with 80-260 mM NaCl gradient in buffer A1.

Transferring:

Article Title: RsgA releases RbfA from 30S ribosome during a late stage of ribosome biosynthesis
Article Snippet: W3110Δ rsgA Δ rbfA was constructed by transferring the kanamycin-resistant marker of W3110Δ rsgA into W3110Δ rbfA via P1 transduction. .. For expression of recombinant RsgA and RbfA, we used E. coli BL21(DE3) (Promega Corporation).

Introduce:

Article Title: The Dengue ED3 Dot Assay, a Novel Serological Test for the Detection of Denguevirus Type-Specific Antibodies and Its Application in a Retrospective Seroprevalence Study
Article Snippet: The forward primers were designed to introduce a BamHI restriction site at the 5′-end and the reverse primers contained a HindIII restriction site at the 3′;-end of the PCR product. .. For expression of the MBP-ED3-fusion proteins, pMAL-p4x plasmids containing the insert of choice were transformed into E. coli BL21(DE3) (Promega, Mannheim, Germany) cells.

Polymerase Chain Reaction:

Article Title: The pH Sensitivity of Murine Heat Shock Protein 47 (HSP47) Binding to Collagen Is Affected by Mutations in the Breach Histidine Cluster *
Article Snippet: The mouse HSP47 cDNA was amplified by PCR using 5′ and 3′ primers with HindIII and NdeI restriction sites, respectively. .. HSP47 WT and HA mutants were expressed in E. coli BL21(DE3)pLysS (Promega, WI).

Article Title: The Dengue ED3 Dot Assay, a Novel Serological Test for the Detection of Denguevirus Type-Specific Antibodies and Its Application in a Retrospective Seroprevalence Study
Article Snippet: After BamHI and HindIII digestion, the PCR fragment was cloned into pMAL-p4x vector (New England Biolabs) downstream to the malE gene [ ]. .. For expression of the MBP-ED3-fusion proteins, pMAL-p4x plasmids containing the insert of choice were transformed into E. coli BL21(DE3) (Promega, Mannheim, Germany) cells.

Article Title: Recombinant N-acyl homoserine lactone-Lactonase AiiAQSI-1 Attenuates Aeromonas hydrophila Virulence Factors, Biofilm Formation and Reduces Mortality in Crucian Carp
Article Snippet: Cloning, Expression, and Purification of AiiAQSI-1 Protein Total genomic DNA from Bacillus sp. strain QSI-1 was extracted using an Ezup Column Bacteria Genomic DNA Purification Kit (Sangon Biotech, Shanghai, China) and used as template for amplification of aiiA gene by PCR. .. The recombinant pET-30a-aiiAQSI-1 vector was then transformed into E. coli BL21(DE3) (Promega, Madison WI,, USA).

Sonication:

Article Title: Identification and in vitro Analysis of the GatD/MurT Enzyme-Complex Catalyzing Lipid II Amidation in Staphylococcus aureusIdentification of Genetic Determinants and Enzymes Involved with the Amidation of Glutamic Acid Residues in the Peptidoglycan of Staphylococcus aureus
Article Snippet: E. coli BL21(DE3) (Promega) cells transformed with the appropriate recombinant plasmid were grown in LB-medium (50 µg/ml ampicillin) at 37°C. .. Aliquots of 200 mg/ml lysozyme, 100 mg/ml DNase and 10 mg/ml RNase were added; cells were incubated for 30 min on ice and sonicated.

Article Title: The Dengue ED3 Dot Assay, a Novel Serological Test for the Detection of Denguevirus Type-Specific Antibodies and Its Application in a Retrospective Seroprevalence Study
Article Snippet: For expression of the MBP-ED3-fusion proteins, pMAL-p4x plasmids containing the insert of choice were transformed into E. coli BL21(DE3) (Promega, Mannheim, Germany) cells. .. Bacterial lysis was carried out by sonication on ice and clear lysates were obtained after centrifugation for 30 min at 4 °C, 15,000× g (Eppendorf 5810R, rotor FA-45-6-30).

Article Title: Recombinant N-acyl homoserine lactone-Lactonase AiiAQSI-1 Attenuates Aeromonas hydrophila Virulence Factors, Biofilm Formation and Reduces Mortality in Crucian Carp
Article Snippet: The recombinant pET-30a-aiiAQSI-1 vector was then transformed into E. coli BL21(DE3) (Promega, Madison WI,, USA). .. After inducible expression with 0.5 mM IPTG at 37 °C for 4 h, cells were harvested and disrupted by sonication.

Binding Assay:

Article Title: The Dengue ED3 Dot Assay, a Novel Serological Test for the Detection of Denguevirus Type-Specific Antibodies and Its Application in a Retrospective Seroprevalence Study
Article Snippet: Paragraph title: 2.4. Maltose Binding Protein-ED3 Fusion Proteins ... For expression of the MBP-ED3-fusion proteins, pMAL-p4x plasmids containing the insert of choice were transformed into E. coli BL21(DE3) (Promega, Mannheim, Germany) cells.

Nucleic Acid Electrophoresis:

Article Title: Recombinant N-acyl homoserine lactone-Lactonase AiiAQSI-1 Attenuates Aeromonas hydrophila Virulence Factors, Biofilm Formation and Reduces Mortality in Crucian Carp
Article Snippet: The recombinant pET-30a-aiiAQSI-1 vector was then transformed into E. coli BL21(DE3) (Promega, Madison WI,, USA). .. The molecular mass of the expressed protein was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).

Mutagenesis:

Article Title: The pH Sensitivity of Murine Heat Shock Protein 47 (HSP47) Binding to Collagen Is Affected by Mutations in the Breach Histidine Cluster *
Article Snippet: Paragraph title: Cloning and Site-directed Mutagenesis ... HSP47 WT and HA mutants were expressed in E. coli BL21(DE3)pLysS (Promega, WI).

Article Title: Glucose-Specific Enzyme IIA of the Phosphoenolpyruvate:Carbohydrate Phosphotransferase System Modulates Chitin Signaling Pathways in Vibrio cholerae
Article Snippet: .. MarC9 (a mutant protein with Himar1 transposase) N-terminally fused to the maltose-binding protein (MBP::MarC9) was purified from E. coli BL21(DE3) (Promega) carrying pMAL-SYMarC9 as described previously ( ). .. By using purified MBP::MarC9, the minitransposon magellan2 (which is a derivative of the Himar1 transposon, encoding Cm resistance) on pEMcat ( ) was introduced into chromosomal DNA from SY0638S.

Isolation:

Article Title: RsgA releases RbfA from 30S ribosome during a late stage of ribosome biosynthesis
Article Snippet: QIG1, QIG4, QIG11, QIG15, QIG26, QIG107, QIG110, QIG121 and QIG128, derivatives of W3110Δ rsgA with restored growth, were isolated in the present study. .. For expression of recombinant RsgA and RbfA, we used E. coli BL21(DE3) (Promega Corporation).

Article Title: Flavonolignans As a Novel Class of Sodium Pump Inhibitors
Article Snippet: .. Expression and purification of the isolated large cytoplasmic segment C45 The large cytoplasmic segment connecting the transmembrane helices 4 and 5 (C45 loop, residues L354-I777 of the mouse brain sequence) with a (His)6 -tag at the N-terminus was expressed in E. coli BL21 (Promega, USA) and purified using a Co2+ -based affinity resin (Clontech, USA) as described previously (Grycova et al., ). ..

Purification:

Article Title: Identification and in vitro Analysis of the GatD/MurT Enzyme-Complex Catalyzing Lipid II Amidation in Staphylococcus aureusIdentification of Genetic Determinants and Enzymes Involved with the Amidation of Glutamic Acid Residues in the Peptidoglycan of Staphylococcus aureus
Article Snippet: Paragraph title: Cloning, overexpression and purification of S. aureus murT , gatD and glnA as His6-tag fusions ... E. coli BL21(DE3) (Promega) cells transformed with the appropriate recombinant plasmid were grown in LB-medium (50 µg/ml ampicillin) at 37°C.

Article Title: Recombinant N-acyl homoserine lactone-Lactonase AiiAQSI-1 Attenuates Aeromonas hydrophila Virulence Factors, Biofilm Formation and Reduces Mortality in Crucian Carp
Article Snippet: Paragraph title: 3.2. Cloning, Expression, and Purification of AiiAQSI-1 Protein ... The recombinant pET-30a-aiiAQSI-1 vector was then transformed into E. coli BL21(DE3) (Promega, Madison WI,, USA).

Article Title: Rare human Caspase-6-R65W and Caspase-6-G66R variants identify a novel regulatory region of Caspase-6 activity
Article Snippet: .. Protein expression and purification Full-length Casp6 was expressed at 30 °C in E. coli BL21(DE3)pLysS (Promega, Fitchburg, WI, USA) and purified using Ni Sepharose Fast Flow 6 (GE Healthcare, Little Chalfont, UK) using Tris buffers pH 8.0. .. Eluted Casp6 was diluted fivefold with buffer A1 (20 mM Tris pH 8.5, 2 mM DTT, 5% glycerol), loaded on Macro Prep High Q resin (Bio-Rad Laboratories, Hercules, CA, USA), washed with 20 mM Tris pH 8.5, 2 mM DTT, 5% glycerol, 50 mM NaCl and eluted with 80-260 mM NaCl gradient in buffer A1.

Article Title: Actin turnover maintains actin filament homeostasis during cytokinetic ring contraction
Article Snippet: .. Preparation of recombinant proteins Recombinant GST-LifeAct-GFP was expressed in Escherichia coli BL21 (DE3-pLysS; Promega) using 0.5 mM IPTG for induction at 30°C for 4 h. The protein was purified on glutathione Sepharose 4B beads according to the manufacturer’s instructions (GE Healthcare). .. The elution buffer containing glutathione was exchanged with reactivation buffer (0.16 M sucrose, 5 mM MgCl2 , 50 mM potassium acetate, and 20 mM MOPS-NaOH, pH 7.0; pH adjusted to 7.5) using PD Minitrap G-10 columns (GE Healthcare).

Article Title: Functional and Biochemical Analysis of Chlamydia trachomatis MurC, an Enzyme Displaying UDP-N-Acetylmuramate:Amino Acid Ligase Activity
Article Snippet: .. For recombinant overexpression and purification of the C. trachomatis MurC domain, plasmid pCTHisSMurC was transformed into E. coli BL21(DE3) (Promega) and grown in 10 liters of Terrific broth ( ) at 37°C until an A 600 of 0.6 was reached. .. Cultures were induced with 1 mM IPTG and grown for a further 3 h before bacteria were harvested by centrifugation.

Article Title: Glucose-Specific Enzyme IIA of the Phosphoenolpyruvate:Carbohydrate Phosphotransferase System Modulates Chitin Signaling Pathways in Vibrio cholerae
Article Snippet: .. MarC9 (a mutant protein with Himar1 transposase) N-terminally fused to the maltose-binding protein (MBP::MarC9) was purified from E. coli BL21(DE3) (Promega) carrying pMAL-SYMarC9 as described previously ( ). .. By using purified MBP::MarC9, the minitransposon magellan2 (which is a derivative of the Himar1 transposon, encoding Cm resistance) on pEMcat ( ) was introduced into chromosomal DNA from SY0638S.

Article Title: Rare human Caspase-6-R65W and Caspase-6-G66R variants identify a novel regulatory region of Caspase-6 activity
Article Snippet: .. Full-length Casp6 was expressed at 30 °C in E. coli BL21(DE3)pLysS (Promega, Fitchburg, WI, USA) and purified using Ni Sepharose Fast Flow 6 (GE Healthcare, Little Chalfont, UK) using Tris buffers pH 8.0. .. Eluted Casp6 was diluted fivefold with buffer A1 (20 mM Tris pH 8.5, 2 mM DTT, 5% glycerol), loaded on Macro Prep High Q resin (Bio-Rad Laboratories, Hercules, CA, USA), washed with 20 mM Tris pH 8.5, 2 mM DTT, 5% glycerol, 50 mM NaCl and eluted with 80-260 mM NaCl gradient in buffer A1.

Article Title: Flavonolignans As a Novel Class of Sodium Pump Inhibitors
Article Snippet: .. Expression and purification of the isolated large cytoplasmic segment C45 The large cytoplasmic segment connecting the transmembrane helices 4 and 5 (C45 loop, residues L354-I777 of the mouse brain sequence) with a (His)6 -tag at the N-terminus was expressed in E. coli BL21 (Promega, USA) and purified using a Co2+ -based affinity resin (Clontech, USA) as described previously (Grycova et al., ). ..

Sequencing:

Article Title: The Dengue ED3 Dot Assay, a Novel Serological Test for the Detection of Denguevirus Type-Specific Antibodies and Its Application in a Retrospective Seroprevalence Study
Article Snippet: Since this gene encodes the malEss signal sequence that directs the maltose binding protein (MBP) to the bacterial periplasm, the reducing environment of the cytoplasm is bypassed to form stable disulfide bonds between the conserved cysteine amino acids present in ED3. .. For expression of the MBP-ED3-fusion proteins, pMAL-p4x plasmids containing the insert of choice were transformed into E. coli BL21(DE3) (Promega, Mannheim, Germany) cells.

Article Title: Flavonolignans As a Novel Class of Sodium Pump Inhibitors
Article Snippet: .. Expression and purification of the isolated large cytoplasmic segment C45 The large cytoplasmic segment connecting the transmembrane helices 4 and 5 (C45 loop, residues L354-I777 of the mouse brain sequence) with a (His)6 -tag at the N-terminus was expressed in E. coli BL21 (Promega, USA) and purified using a Co2+ -based affinity resin (Clontech, USA) as described previously (Grycova et al., ). ..

Article Title: Physical Interaction between Coat Morphogenetic Proteins SpoVID and CotE Is Necessary for Spore Encasement in Bacillus subtilis
Article Snippet: E. coli BL21(DE3) (Promega) was used for expression of cotE-STOP and spoVID-his constructs (see below). .. The coding sequence of spoVID was cloned into the vector using the NdeI and XhoI sites, creating, when expressed, an in-frame His6 tag fusion to the C terminus of SpoVID.

Selection:

Article Title: Glucose-Specific Enzyme IIA of the Phosphoenolpyruvate:Carbohydrate Phosphotransferase System Modulates Chitin Signaling Pathways in Vibrio cholerae
Article Snippet: Paragraph title: Transposon mutagenesis, mutant selection, and insertion site mapping. ... MarC9 (a mutant protein with Himar1 transposase) N-terminally fused to the maltose-binding protein (MBP::MarC9) was purified from E. coli BL21(DE3) (Promega) carrying pMAL-SYMarC9 as described previously ( ).

Staining:

Article Title: Flavonolignans As a Novel Class of Sodium Pump Inhibitors
Article Snippet: Expression and purification of the isolated large cytoplasmic segment C45 The large cytoplasmic segment connecting the transmembrane helices 4 and 5 (C45 loop, residues L354-I777 of the mouse brain sequence) with a (His)6 -tag at the N-terminus was expressed in E. coli BL21 (Promega, USA) and purified using a Co2+ -based affinity resin (Clontech, USA) as described previously (Grycova et al., ). .. Protein were analyzed by Coomassie blue stained SDS PAGE and concentration was determined using the Bradford assay (Bradford, ) using BSA as a standard.

SDS Page:

Article Title: Recombinant N-acyl homoserine lactone-Lactonase AiiAQSI-1 Attenuates Aeromonas hydrophila Virulence Factors, Biofilm Formation and Reduces Mortality in Crucian Carp
Article Snippet: The recombinant pET-30a-aiiAQSI-1 vector was then transformed into E. coli BL21(DE3) (Promega, Madison WI,, USA). .. The molecular mass of the expressed protein was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).

Article Title: Identification and in vitro Analysis of the GatD/MurT Enzyme-Complex Catalyzing Lipid II Amidation in Staphylococcus aureusIdentification of Genetic Determinants and Enzymes Involved with the Amidation of Glutamic Acid Residues in the Peptidoglycan of Staphylococcus aureus
Article Snippet: The corresponding plasmid was introduced into E. coli BL21 (Promega) and overexpression was carried out as described above. .. Co-elution from Ni-NTA column (Qiagen) of the GatD-His6 /MurT complex was analyzed by SDS page.

Article Title: Flavonolignans As a Novel Class of Sodium Pump Inhibitors
Article Snippet: Expression and purification of the isolated large cytoplasmic segment C45 The large cytoplasmic segment connecting the transmembrane helices 4 and 5 (C45 loop, residues L354-I777 of the mouse brain sequence) with a (His)6 -tag at the N-terminus was expressed in E. coli BL21 (Promega, USA) and purified using a Co2+ -based affinity resin (Clontech, USA) as described previously (Grycova et al., ). .. Protein were analyzed by Coomassie blue stained SDS PAGE and concentration was determined using the Bradford assay (Bradford, ) using BSA as a standard.

Plasmid Preparation:

Article Title: A male sterility-associated cytotoxic protein ORF288 in Brassica juncea causes aborted pollen development
Article Snippet: The corresponding enzymes were then used to clone this fragment into the bacterial expression vector PET32a (Novagen). .. The expression of ORF288 in E. coli BL21(DE3)plysS (Promega) was induced by adding 0.5 mM isopropyl-β-D -thiogalactopyranoside (IPTG) when the optical density (OD) of the samples reached ∼0.6.

Article Title: Identification and in vitro Analysis of the GatD/MurT Enzyme-Complex Catalyzing Lipid II Amidation in Staphylococcus aureusIdentification of Genetic Determinants and Enzymes Involved with the Amidation of Glutamic Acid Residues in the Peptidoglycan of Staphylococcus aureus
Article Snippet: .. E. coli BL21(DE3) (Promega) cells transformed with the appropriate recombinant plasmid were grown in LB-medium (50 µg/ml ampicillin) at 37°C. .. At an OD600 of 0.6, IPTG was added at a concentration of 0.75 mM to induce expression of the recombinant proteins.

Article Title: Identification of a Cytoplasmic Complex That Adds a Cap onto 5?-Monophosphate RNA ▿Identification of a Cytoplasmic Complex That Adds a Cap onto 5?-Monophosphate RNA ▿ §
Article Snippet: .. Escherichia coli BL21(DE3)pLysS (Promega) was transformed with the plasmid pGEX-meIF4E, expressing a glutathione S -transferase (GST) fusion protein with murine eIF4E cap-binding protein (GST-eIF4E). .. Cleared bacterial lysate was incubated with m7 GTP-Sepharose (GE Biosciences) by end-over-end rotation at 4°C overnight.

Article Title: The pH Sensitivity of Murine Heat Shock Protein 47 (HSP47) Binding to Collagen Is Affected by Mutations in the Breach Histidine Cluster *
Article Snippet: Subsequently, the gene was cloned into the pET-24b(+) vector (Novagen, Germany), which contains the equivalent restriction sites. .. HSP47 WT and HA mutants were expressed in E. coli BL21(DE3)pLysS (Promega, WI).

Article Title: The Dengue ED3 Dot Assay, a Novel Serological Test for the Detection of Denguevirus Type-Specific Antibodies and Its Application in a Retrospective Seroprevalence Study
Article Snippet: After BamHI and HindIII digestion, the PCR fragment was cloned into pMAL-p4x vector (New England Biolabs) downstream to the malE gene [ ]. .. For expression of the MBP-ED3-fusion proteins, pMAL-p4x plasmids containing the insert of choice were transformed into E. coli BL21(DE3) (Promega, Mannheim, Germany) cells.

Article Title: Recombinant N-acyl homoserine lactone-Lactonase AiiAQSI-1 Attenuates Aeromonas hydrophila Virulence Factors, Biofilm Formation and Reduces Mortality in Crucian Carp
Article Snippet: .. The recombinant pET-30a-aiiAQSI-1 vector was then transformed into E. coli BL21(DE3) (Promega, Madison WI,, USA). .. After inducible expression with 0.5 mM IPTG at 37 °C for 4 h, cells were harvested and disrupted by sonication.

Article Title: Identification and in vitro Analysis of the GatD/MurT Enzyme-Complex Catalyzing Lipid II Amidation in Staphylococcus aureusIdentification of Genetic Determinants and Enzymes Involved with the Amidation of Glutamic Acid Residues in the Peptidoglycan of Staphylococcus aureus
Article Snippet: .. The corresponding plasmid was introduced into E. coli BL21 (Promega) and overexpression was carried out as described above. .. Co-elution from Ni-NTA column (Qiagen) of the GatD-His6 /MurT complex was analyzed by SDS page.

Article Title: Functional and Biochemical Analysis of Chlamydia trachomatis MurC, an Enzyme Displaying UDP-N-Acetylmuramate:Amino Acid Ligase Activity
Article Snippet: .. For recombinant overexpression and purification of the C. trachomatis MurC domain, plasmid pCTHisSMurC was transformed into E. coli BL21(DE3) (Promega) and grown in 10 liters of Terrific broth ( ) at 37°C until an A 600 of 0.6 was reached. .. Cultures were induced with 1 mM IPTG and grown for a further 3 h before bacteria were harvested by centrifugation.

Article Title: RsgA releases RbfA from 30S ribosome during a late stage of ribosome biosynthesis
Article Snippet: For expression of recombinant RsgA and RbfA, we used E. coli BL21(DE3) (Promega Corporation). .. Cultures of constructed strains were grown at 37°C in LB medium with agitation; ampicillin (50 μg/ml) was added to the medium for strains harbouring a plasmid with an ampicillin-resistant marker (pUC26-2, pMW118, pMWrsgA, pMWrbfA+ or its variants and pGEMrbfA or its variants), and chloramphenicol (10 μg/ml) was added to the medium for strains harbouring a plasmid with a chloramphenicol-resistant marker (pCA24N– and pCArbfA).

Article Title: Physical Interaction between Coat Morphogenetic Proteins SpoVID and CotE Is Necessary for Spore Encasement in Bacillus subtilis
Article Snippet: E. coli BL21(DE3) (Promega) was used for expression of cotE-STOP and spoVID-his constructs (see below). .. For the His6 tag pulldown assays, both cotE-STOP and spoVID-his constructs were designed using the pET21b vector (EMD4Biosciences).

Copurification:

Article Title: Identification and in vitro Analysis of the GatD/MurT Enzyme-Complex Catalyzing Lipid II Amidation in Staphylococcus aureusIdentification of Genetic Determinants and Enzymes Involved with the Amidation of Glutamic Acid Residues in the Peptidoglycan of Staphylococcus aureus
Article Snippet: Paragraph title: Co-purification of the GatD/MurT enzyme complex ... The corresponding plasmid was introduced into E. coli BL21 (Promega) and overexpression was carried out as described above.

Co-Elution Assay:

Article Title: Identification and in vitro Analysis of the GatD/MurT Enzyme-Complex Catalyzing Lipid II Amidation in Staphylococcus aureusIdentification of Genetic Determinants and Enzymes Involved with the Amidation of Glutamic Acid Residues in the Peptidoglycan of Staphylococcus aureus
Article Snippet: The corresponding plasmid was introduced into E. coli BL21 (Promega) and overexpression was carried out as described above. .. Co-elution from Ni-NTA column (Qiagen) of the GatD-His6 /MurT complex was analyzed by SDS page.

Spectrophotometry:

Article Title: A male sterility-associated cytotoxic protein ORF288 in Brassica juncea causes aborted pollen development
Article Snippet: The expression of ORF288 in E. coli BL21(DE3)plysS (Promega) was induced by adding 0.5 mM isopropyl-β-D -thiogalactopyranoside (IPTG) when the optical density (OD) of the samples reached ∼0.6. .. Over the next 2.5 h, the OD of the samples was measured every 30 min at 600 nm using a UV-1601 spectrophotometer (Shimadzu, Japan)

Concentration Assay:

Article Title: Identification and in vitro Analysis of the GatD/MurT Enzyme-Complex Catalyzing Lipid II Amidation in Staphylococcus aureusIdentification of Genetic Determinants and Enzymes Involved with the Amidation of Glutamic Acid Residues in the Peptidoglycan of Staphylococcus aureus
Article Snippet: E. coli BL21(DE3) (Promega) cells transformed with the appropriate recombinant plasmid were grown in LB-medium (50 µg/ml ampicillin) at 37°C. .. At an OD600 of 0.6, IPTG was added at a concentration of 0.75 mM to induce expression of the recombinant proteins.

Article Title: Flavonolignans As a Novel Class of Sodium Pump Inhibitors
Article Snippet: Expression and purification of the isolated large cytoplasmic segment C45 The large cytoplasmic segment connecting the transmembrane helices 4 and 5 (C45 loop, residues L354-I777 of the mouse brain sequence) with a (His)6 -tag at the N-terminus was expressed in E. coli BL21 (Promega, USA) and purified using a Co2+ -based affinity resin (Clontech, USA) as described previously (Grycova et al., ). .. Protein were analyzed by Coomassie blue stained SDS PAGE and concentration was determined using the Bradford assay (Bradford, ) using BSA as a standard.

CTG Assay:

Article Title: The pH Sensitivity of Murine Heat Shock Protein 47 (HSP47) Binding to Collagen Is Affected by Mutations in the Breach Histidine Cluster *
Article Snippet: The primers used were as follows (with the introduced mutation sites underlined ): H191A, 5′-ATG TTC TTT AAG CCA GCA TGG GAT GAG AAG TTT; H197A, 5′-CA CAC TGG GAT GAG AAG TTT CAC GCA AAG ATG GTG G; H198A, 5′-CA CAC TGG GAT GAG AAG TTT GCA CAC AAG ATG GTG G; H197A,H198A, 5′-CA CAC TGG GAT GAG AAG TTT GCA GCA AAG ATG GTG G; H244A, 5′-G ATG CCC CTG GCT GCA AAG CTC TCC AGC; H255A,H256A, 5′-GC CTC ATC ATC CTC ATG CCC GCA GCA GTG GAG CCG C; H302A, 5′-CAT GAC CTG CAG AAA GCC CTG GCA GGA; W110F, 5′-ACT GCG CGC AAC GTG ACC TTT AAA CTG GGC AGC CGC; W158F, 5′-TCC ATC AAC GAG TTT GCC TCG CAG ACC ACG; W192F, 5′-TTC TTT AAG CCA CAC TTT GAT GAG AAG TTT CAG; W275F, 5′-GAG CAG CTG AAG GCC TTT ATG GGA AAG ATG CAG AAG; W341F, 5′-ACT GCC TTC GAG TTC GAC ACC GAG GGC AAC. .. HSP47 WT and HA mutants were expressed in E. coli BL21(DE3)pLysS (Promega, WI).

DNA Purification:

Article Title: Recombinant N-acyl homoserine lactone-Lactonase AiiAQSI-1 Attenuates Aeromonas hydrophila Virulence Factors, Biofilm Formation and Reduces Mortality in Crucian Carp
Article Snippet: Cloning, Expression, and Purification of AiiAQSI-1 Protein Total genomic DNA from Bacillus sp. strain QSI-1 was extracted using an Ezup Column Bacteria Genomic DNA Purification Kit (Sangon Biotech, Shanghai, China) and used as template for amplification of aiiA gene by PCR. .. The recombinant pET-30a-aiiAQSI-1 vector was then transformed into E. coli BL21(DE3) (Promega, Madison WI,, USA).

Lysis:

Article Title: Identification and in vitro Analysis of the GatD/MurT Enzyme-Complex Catalyzing Lipid II Amidation in Staphylococcus aureusIdentification of Genetic Determinants and Enzymes Involved with the Amidation of Glutamic Acid Residues in the Peptidoglycan of Staphylococcus aureus
Article Snippet: E. coli BL21(DE3) (Promega) cells transformed with the appropriate recombinant plasmid were grown in LB-medium (50 µg/ml ampicillin) at 37°C. .. After 4 h, cells were harvested and resuspended in lysis buffer (50 mM Tris/HCl, pH 7.5, 300 mM NaCl, 10 mM imidazole).

Article Title: The Dengue ED3 Dot Assay, a Novel Serological Test for the Detection of Denguevirus Type-Specific Antibodies and Its Application in a Retrospective Seroprevalence Study
Article Snippet: For expression of the MBP-ED3-fusion proteins, pMAL-p4x plasmids containing the insert of choice were transformed into E. coli BL21(DE3) (Promega, Mannheim, Germany) cells. .. The bacteria were harvested after four hours via centrifugation at 10,000 rpm for 3 min (Beckman Coulter Avanti J-26 XP, rotor JA-14) and were suspended in lysis buffer (20 mM Tris pH 7.4, 200 mM NaCl, 1 mM EDTA, 5 mg/mL lysozyme).

Article Title: Functional and Biochemical Analysis of Chlamydia trachomatis MurC, an Enzyme Displaying UDP-N-Acetylmuramate:Amino Acid Ligase Activity
Article Snippet: For recombinant overexpression and purification of the C. trachomatis MurC domain, plasmid pCTHisSMurC was transformed into E. coli BL21(DE3) (Promega) and grown in 10 liters of Terrific broth ( ) at 37°C until an A 600 of 0.6 was reached. .. The cell pellet was resuspended in 100 ml of lysis buffer (50 mM sodium phosphate [pH 8], 300 mM NaCl, 5 mM 2-mercaptoethanol) containing 10 mM imidazole.

Marker:

Article Title: RsgA releases RbfA from 30S ribosome during a late stage of ribosome biosynthesis
Article Snippet: W3110Δ rsgA Δ rbfA was constructed by transferring the kanamycin-resistant marker of W3110Δ rsgA into W3110Δ rbfA via P1 transduction. .. For expression of recombinant RsgA and RbfA, we used E. coli BL21(DE3) (Promega Corporation).

Recombinant:

Article Title: Identification and in vitro Analysis of the GatD/MurT Enzyme-Complex Catalyzing Lipid II Amidation in Staphylococcus aureusIdentification of Genetic Determinants and Enzymes Involved with the Amidation of Glutamic Acid Residues in the Peptidoglycan of Staphylococcus aureus
Article Snippet: .. E. coli BL21(DE3) (Promega) cells transformed with the appropriate recombinant plasmid were grown in LB-medium (50 µg/ml ampicillin) at 37°C. .. At an OD600 of 0.6, IPTG was added at a concentration of 0.75 mM to induce expression of the recombinant proteins.

Article Title: Recombinant N-acyl homoserine lactone-Lactonase AiiAQSI-1 Attenuates Aeromonas hydrophila Virulence Factors, Biofilm Formation and Reduces Mortality in Crucian Carp
Article Snippet: .. The recombinant pET-30a-aiiAQSI-1 vector was then transformed into E. coli BL21(DE3) (Promega, Madison WI,, USA). .. After inducible expression with 0.5 mM IPTG at 37 °C for 4 h, cells were harvested and disrupted by sonication.

Article Title: Actin turnover maintains actin filament homeostasis during cytokinetic ring contraction
Article Snippet: .. Preparation of recombinant proteins Recombinant GST-LifeAct-GFP was expressed in Escherichia coli BL21 (DE3-pLysS; Promega) using 0.5 mM IPTG for induction at 30°C for 4 h. The protein was purified on glutathione Sepharose 4B beads according to the manufacturer’s instructions (GE Healthcare). .. The elution buffer containing glutathione was exchanged with reactivation buffer (0.16 M sucrose, 5 mM MgCl2 , 50 mM potassium acetate, and 20 mM MOPS-NaOH, pH 7.0; pH adjusted to 7.5) using PD Minitrap G-10 columns (GE Healthcare).

Article Title: Functional and Biochemical Analysis of Chlamydia trachomatis MurC, an Enzyme Displaying UDP-N-Acetylmuramate:Amino Acid Ligase Activity
Article Snippet: .. For recombinant overexpression and purification of the C. trachomatis MurC domain, plasmid pCTHisSMurC was transformed into E. coli BL21(DE3) (Promega) and grown in 10 liters of Terrific broth ( ) at 37°C until an A 600 of 0.6 was reached. .. Cultures were induced with 1 mM IPTG and grown for a further 3 h before bacteria were harvested by centrifugation.

Article Title: RsgA releases RbfA from 30S ribosome during a late stage of ribosome biosynthesis
Article Snippet: .. For expression of recombinant RsgA and RbfA, we used E. coli BL21(DE3) (Promega Corporation). .. Cultures of constructed strains were grown at 37°C in LB medium with agitation; ampicillin (50 μg/ml) was added to the medium for strains harbouring a plasmid with an ampicillin-resistant marker (pUC26-2, pMW118, pMWrsgA, pMWrbfA+ or its variants and pGEMrbfA or its variants), and chloramphenicol (10 μg/ml) was added to the medium for strains harbouring a plasmid with a chloramphenicol-resistant marker (pCA24N– and pCArbfA).

Positron Emission Tomography:

Article Title: The pH Sensitivity of Murine Heat Shock Protein 47 (HSP47) Binding to Collagen Is Affected by Mutations in the Breach Histidine Cluster *
Article Snippet: Subsequently, the gene was cloned into the pET-24b(+) vector (Novagen, Germany), which contains the equivalent restriction sites. .. HSP47 WT and HA mutants were expressed in E. coli BL21(DE3)pLysS (Promega, WI).

Article Title: Recombinant N-acyl homoserine lactone-Lactonase AiiAQSI-1 Attenuates Aeromonas hydrophila Virulence Factors, Biofilm Formation and Reduces Mortality in Crucian Carp
Article Snippet: .. The recombinant pET-30a-aiiAQSI-1 vector was then transformed into E. coli BL21(DE3) (Promega, Madison WI,, USA). .. After inducible expression with 0.5 mM IPTG at 37 °C for 4 h, cells were harvested and disrupted by sonication.

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