bl21 startm de3  (Thermo Fisher)


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    Structured Review

    Thermo Fisher bl21 startm de3
    Bl21 Startm De3, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 88/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/bl21 startm de3/product/Thermo Fisher
    Average 88 stars, based on 4 article reviews
    Price from $9.99 to $1999.99
    bl21 startm de3 - by Bioz Stars, 2020-02
    88/100 stars

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    Related Articles

    Clone Assay:

    Article Title: A (–)-kolavenyl diphosphate synthase catalyzes the first step of salvinorin A biosynthesis in Salvia divinorum
    Article Snippet: Paragraph title: Cloning and heterologous expression of full-length and truncated SdKPS and its mutants ... The expression of recombinant proteins was carried out in BL21 StarTM (DE3) (ThermoFisher) cells induced via the auto-induction method ( ) at 18 °C to 20 °C.

    Article Title: Insights into an evolutionary strategy leading to antibiotic resistance
    Article Snippet: .. The resulting PCR products were cloned into the pET26b(+) vector, and the constructs were then transferred into BL21 StarTM(DE3) (Invitrogen, California, USA). .. Subsequently, the transformed cells were grown on Mueller-Hinton agar plates, supplemented with 50 μg/mL kanamycin and 50–125 μg/mL cefoxitin for selection.

    Article Title: Characterization of Conformation-dependent Prion Protein Epitopes *
    Article Snippet: Selected shuffled DNA constructs were PCR-amplified and cloned into pET100/D-TOPO® (Invitrogen). .. Transformed BL21 StarTM (DE3) (Invitrogen) were cultured until the absorbance value reached 1.0 at 650 nm and induced using isopropyl 1-thio-β- d -galactopyranoside (Sigma) at a final concentration of 1 m m .

    Article Title: Interacting Proteins on Human Spermatozoa: Adaptive Evolution of the Binding of Semenogelin I to EPPIN
    Article Snippet: Additionally, C-terminal and N-terminal truncated fragments flanking the residue Cys239 from the SEMG1 primary sequence, corresponding to residues R165-R281 (SEMG174-42 ), R165-Q247 (SEMG174-8 ), Q207-R281 (SEMG132-42 ), Y220-L262 (SEMG119-22 ), E229-L269 (SEMG110-30 ), and Q207-Q247 (SEMG132-8 ) ( ) were produced and cloned into the same vector. .. Recombinant proteins were expressed either in BL21 StarTM (DE3) One Shot® E. coli (Invitrogen; SEMG1 constructs) or E. coli Rosetta-Gami 2TM (DE3) (Novagen, Madison, WI; EPPIN construct) and purified under denaturing conditions using nickel-nitrilotriacetic acid agarose (Ni-NTA) beads (Qiagen, Valencia, CA) as described previously [ ].

    Article Title: The RNase R from Campylobacter jejuni Has Unique Features and Is Involved in the First Steps of Infection *
    Article Snippet: We have cloned the rnr gene from C. jejuni in an expression vector. .. We tested several conditions for protein expression in different expression strains, and finally, we expressed Cj -RNR at 32 °C in a BL21 StarTM (DE3) (Invitrogen).

    Article Title: Kif2C Minimal Functional Domain Has Unusual Nucleotide Binding Properties That Are Adapted to Microtubule Depolymerization *
    Article Snippet: The HsKif2C cDNA was purchased from Invitrogen, and fragments coding for Kif2C-(N+M) (Ser-187–Gln-598), Kif2C-(sN+M) (Arg-216–Gln-598), and Kif2C-Motor (Asp-252–Gln-598) ( A ) were amplified by PCR and cloned into the NcoI and XhoI pET28a restriction sites for expression. .. All the recombinant Kif2C fragments were expressed in BL21 StarTM (DE3) (Invitrogen) and purified on a HisTrap FF column followed by a Mono S column (GE Healthcare).

    Centrifugation:

    Article Title: Protein Methionine Sulfoxide Dynamics in Arabidopsis thaliana
    Article Snippet: BL21 StarTM One Shot chemically competent Escherichia coli cells (Invitrogen) were transformed, and GSTF9 and GSTT23 protein production was induced with 0.2 m m isopropyl β- d -1-thiogalactopyranoside in growing cultures of successfully transformed E. coli cells reaching an A 600 of 0.8. .. Sonication for 5 min with 10-s pulses was followed by centrifugation for 10 min at 10,000 × g to remove cellular debris after which the recombinant GST proteins were purified from the supernatant by affinity chromatography using immobilized glutathione columns (GSTrap HP, GE Healthcare).

    Article Title: A (–)-kolavenyl diphosphate synthase catalyzes the first step of salvinorin A biosynthesis in Salvia divinorum
    Article Snippet: The expression of recombinant proteins was carried out in BL21 StarTM (DE3) (ThermoFisher) cells induced via the auto-induction method ( ) at 18 °C to 20 °C. .. The supernatant after centrifugation (20 000 g , 4 °C, 10 min) was loaded to a pre-equilibrated nickel-nitrilotriacetic acid agarose matrix (Qiagen).

    Article Title: Allergen valency, dose and FcεRI occupancy set thresholds for secretory responses to Pen a 1 and motivate design of hypoallergens
    Article Snippet: .. All recombinant proteins were expressed in BL21 starTM(DE3) cells (Invitrogen, Grand Island, NY) induced with 1mM isopropylthio-β-galactoside for 4 h. Cells were harvested by centrifugation and stored at −80°C overnight. ..

    Amplification:

    Article Title: A (–)-kolavenyl diphosphate synthase catalyzes the first step of salvinorin A biosynthesis in Salvia divinorum
    Article Snippet: Cloning and heterologous expression of full-length and truncated SdKPS and its mutants The open reading frames of full-length (Fl) and truncated (Tr) SdKPS were obtained with primers that amplified cDNA from the targeted sites (SdKPS-FULL-F and SdKPS-exp-R for Fl-SdKPS, SdKPS-truncation-F and SdKPS-exp-R for Tr-SdKPS; for details see Supplementary Table S1 at JXB online), followed by transfer into the pCR2.1 TOPO TA cloning vector (Invitrogen), and verification by complete sequencing. .. The expression of recombinant proteins was carried out in BL21 StarTM (DE3) (ThermoFisher) cells induced via the auto-induction method ( ) at 18 °C to 20 °C.

    Article Title: Adenine Aminohydrolase from Leishmania donovani
    Article Snippet: The AAH ORF was amplified by PCR from the AAH cosmid, ligated into the pET200/D-TOPO® bacterial expression vector (Invitrogen), which automatically attaches a His6 tag to the NH2 terminus of the inserted gene product, and sequenced to ensure fidelity of the construct. .. The pET200/D-TOPO®- AAH construct was then transformed into BL21 StarTM (DE3) One Shot® Escherichia coli (Invitrogen).

    Article Title: Kif2C Minimal Functional Domain Has Unusual Nucleotide Binding Properties That Are Adapted to Microtubule Depolymerization *
    Article Snippet: The HsKif2C cDNA was purchased from Invitrogen, and fragments coding for Kif2C-(N+M) (Ser-187–Gln-598), Kif2C-(sN+M) (Arg-216–Gln-598), and Kif2C-Motor (Asp-252–Gln-598) ( A ) were amplified by PCR and cloned into the NcoI and XhoI pET28a restriction sites for expression. .. All the recombinant Kif2C fragments were expressed in BL21 StarTM (DE3) (Invitrogen) and purified on a HisTrap FF column followed by a Mono S column (GE Healthcare).

    TA Cloning:

    Article Title: A (–)-kolavenyl diphosphate synthase catalyzes the first step of salvinorin A biosynthesis in Salvia divinorum
    Article Snippet: Cloning and heterologous expression of full-length and truncated SdKPS and its mutants The open reading frames of full-length (Fl) and truncated (Tr) SdKPS were obtained with primers that amplified cDNA from the targeted sites (SdKPS-FULL-F and SdKPS-exp-R for Fl-SdKPS, SdKPS-truncation-F and SdKPS-exp-R for Tr-SdKPS; for details see Supplementary Table S1 at JXB online), followed by transfer into the pCR2.1 TOPO TA cloning vector (Invitrogen), and verification by complete sequencing. .. The expression of recombinant proteins was carried out in BL21 StarTM (DE3) (ThermoFisher) cells induced via the auto-induction method ( ) at 18 °C to 20 °C.

    Construct:

    Article Title: Insights into an evolutionary strategy leading to antibiotic resistance
    Article Snippet: .. The resulting PCR products were cloned into the pET26b(+) vector, and the constructs were then transferred into BL21 StarTM(DE3) (Invitrogen, California, USA). .. Subsequently, the transformed cells were grown on Mueller-Hinton agar plates, supplemented with 50 μg/mL kanamycin and 50–125 μg/mL cefoxitin for selection.

    Article Title: Characterization of Conformation-dependent Prion Protein Epitopes *
    Article Snippet: Selected shuffled DNA constructs were PCR-amplified and cloned into pET100/D-TOPO® (Invitrogen). .. Transformed BL21 StarTM (DE3) (Invitrogen) were cultured until the absorbance value reached 1.0 at 650 nm and induced using isopropyl 1-thio-β- d -galactopyranoside (Sigma) at a final concentration of 1 m m .

    Article Title: Interacting Proteins on Human Spermatozoa: Adaptive Evolution of the Binding of Semenogelin I to EPPIN
    Article Snippet: .. Recombinant proteins were expressed either in BL21 StarTM (DE3) One Shot® E. coli (Invitrogen; SEMG1 constructs) or E. coli Rosetta-Gami 2TM (DE3) (Novagen, Madison, WI; EPPIN construct) and purified under denaturing conditions using nickel-nitrilotriacetic acid agarose (Ni-NTA) beads (Qiagen, Valencia, CA) as described previously [ ]. .. Removal of N-terminal 6X His-tag from recombinant EPPIN was performed using recombinant enterokinase cleavage/capture kit (Novagen), according to the manufacturer’s instructions.

    Article Title: Adenine Aminohydrolase from Leishmania donovani
    Article Snippet: .. The pET200/D-TOPO®- AAH construct was then transformed into BL21 StarTM (DE3) One Shot® Escherichia coli (Invitrogen). .. After induction in 0.5 m m isopropyl 1-thio-β- d -galactopyranoside, the recombinant L. donovani AAH protein was purified to virtual homogeneity over a Ni2+ -NTA-agarose column from E. coli extracts that had been prepared using a French press as described previously ( ).

    Article Title: Kif2C Minimal Functional Domain Has Unusual Nucleotide Binding Properties That Are Adapted to Microtubule Depolymerization *
    Article Snippet: Paragraph title: Constructs and Protein Purification ... All the recombinant Kif2C fragments were expressed in BL21 StarTM (DE3) (Invitrogen) and purified on a HisTrap FF column followed by a Mono S column (GE Healthcare).

    Incubation:

    Article Title: Protein Methionine Sulfoxide Dynamics in Arabidopsis thaliana
    Article Snippet: BL21 StarTM One Shot chemically competent Escherichia coli cells (Invitrogen) were transformed, and GSTF9 and GSTT23 protein production was induced with 0.2 m m isopropyl β- d -1-thiogalactopyranoside in growing cultures of successfully transformed E. coli cells reaching an A 600 of 0.8. .. After incubation overnight at 16 °C, cells were centrifuged for 10 min at 2700 × g and resuspended in 20 ml of cold PBS with 1 m m DTT, 1% Triton X-100, and the appropriate amount of Complete protease inhibitor mixture.

    Article Title: A (–)-kolavenyl diphosphate synthase catalyzes the first step of salvinorin A biosynthesis in Salvia divinorum
    Article Snippet: The expression of recombinant proteins was carried out in BL21 StarTM (DE3) (ThermoFisher) cells induced via the auto-induction method ( ) at 18 °C to 20 °C. .. The slurry was incubated with mild agitation at 4 °C for 1 h and then was washed with 10 volumes of washing buffer (20 mM imidazole).

    Article Title: Interacting Proteins on Human Spermatozoa: Adaptive Evolution of the Binding of Semenogelin I to EPPIN
    Article Snippet: Recombinant proteins were expressed either in BL21 StarTM (DE3) One Shot® E. coli (Invitrogen; SEMG1 constructs) or E. coli Rosetta-Gami 2TM (DE3) (Novagen, Madison, WI; EPPIN construct) and purified under denaturing conditions using nickel-nitrilotriacetic acid agarose (Ni-NTA) beads (Qiagen, Valencia, CA) as described previously [ ]. .. For removal of undigested 6X His-tag-EPPIN, the cleavage reaction was incubated with Ni-NTA beads at room temperature for 30 min with gentle mixing and tag-free EPPIN was then eluted in PBS buffer containing 1 M urea.

    Activity Assay:

    Article Title: The RNase R from Campylobacter jejuni Has Unique Features and Is Involved in the First Steps of Infection *
    Article Snippet: Paragraph title: Cj-RNR Activity at Different Conditions ... We tested several conditions for protein expression in different expression strains, and finally, we expressed Cj -RNR at 32 °C in a BL21 StarTM (DE3) (Invitrogen).

    Article Title: Kif2C Minimal Functional Domain Has Unusual Nucleotide Binding Properties That Are Adapted to Microtubule Depolymerization *
    Article Snippet: R330A and R379A mutations, which do not affect the Kif2C activity , were introduced in all the constructs to diminish protein aggregation in the presence of tubulin. .. All the recombinant Kif2C fragments were expressed in BL21 StarTM (DE3) (Invitrogen) and purified on a HisTrap FF column followed by a Mono S column (GE Healthcare).

    Expressing:

    Article Title: Protein Methionine Sulfoxide Dynamics in Arabidopsis thaliana
    Article Snippet: Paragraph title: Recombinant GST Protein Expression and Purification ... BL21 StarTM One Shot chemically competent Escherichia coli cells (Invitrogen) were transformed, and GSTF9 and GSTT23 protein production was induced with 0.2 m m isopropyl β- d -1-thiogalactopyranoside in growing cultures of successfully transformed E. coli cells reaching an A 600 of 0.8.

    Article Title: A (–)-kolavenyl diphosphate synthase catalyzes the first step of salvinorin A biosynthesis in Salvia divinorum
    Article Snippet: .. The expression of recombinant proteins was carried out in BL21 StarTM (DE3) (ThermoFisher) cells induced via the auto-induction method ( ) at 18 °C to 20 °C. .. Pelleted cells were suspended in binding buffer (20 mM HEPES, pH 7.5, 500 mM NaCl, 5 mM imidazole, and 5% glycerol) containing 0.01% (w/v) lysozyme and then disrupted by sonication.

    Article Title: Insights into an evolutionary strategy leading to antibiotic resistance
    Article Snippet: The resulting PCR products were cloned into the pET26b(+) vector, and the constructs were then transferred into BL21 StarTM(DE3) (Invitrogen, California, USA). .. To ensure a low expression level a final concentration of 50 μM isopropyl-β-D-1- thiogalactopyranoside (IPTG) (Solon, OH, USA) was used in all selection media.

    Article Title: Interacting Proteins on Human Spermatozoa: Adaptive Evolution of the Binding of Semenogelin I to EPPIN
    Article Snippet: Paragraph title: Production and Expression of Recombinant Proteins ... Recombinant proteins were expressed either in BL21 StarTM (DE3) One Shot® E. coli (Invitrogen; SEMG1 constructs) or E. coli Rosetta-Gami 2TM (DE3) (Novagen, Madison, WI; EPPIN construct) and purified under denaturing conditions using nickel-nitrilotriacetic acid agarose (Ni-NTA) beads (Qiagen, Valencia, CA) as described previously [ ].

    Article Title: Adenine Aminohydrolase from Leishmania donovani
    Article Snippet: Paragraph title: AAH Expression and Purification ... The pET200/D-TOPO®- AAH construct was then transformed into BL21 StarTM (DE3) One Shot® Escherichia coli (Invitrogen).

    Article Title: Allergen valency, dose and FcεRI occupancy set thresholds for secretory responses to Pen a 1 and motivate design of hypoallergens
    Article Snippet: Paragraph title: Expression and Purification of Recombinant Allergens ... All recombinant proteins were expressed in BL21 starTM(DE3) cells (Invitrogen, Grand Island, NY) induced with 1mM isopropylthio-β-galactoside for 4 h. Cells were harvested by centrifugation and stored at −80°C overnight.

    Article Title: A Versatile Strategy for Production of Membrane Proteins with Diverse Topologies: Application to Investigation of Bacterial Homologues of Human Divalent Metal Ion and Nucleoside Transporters
    Article Snippet: .. Expression trials Expression trials were typically performed in four E . coli strains in parallel: BL21-gold(DE3) (Stratagene), BL21 StarTM (DE3) (Invitrogen; [ ]), C41(DE3) and C43(DE3) (Lucigen Corporation; [ ]), all of which had been transformed with the plasmid pRARE2 (Novagen) to enhance translation efficiency of ORFs with codon usage different from endogenous E . coli genes [ ]. .. Typically, autoinduction of 1 mL cultures in modified M9 medium (M9auto ); Lysogeny broth (LBauto ) or Superbroth medium (SBauto ) containing 0.5% glycerol, 0.05% glucose and 0.2% lactose was performed at 37°C for 24 h in 24-deep-well plates as previously described [ ].

    Article Title: The RNase R from Campylobacter jejuni Has Unique Features and Is Involved in the First Steps of Infection *
    Article Snippet: .. We tested several conditions for protein expression in different expression strains, and finally, we expressed Cj -RNR at 32 °C in a BL21 StarTM (DE3) (Invitrogen). .. We then purified the protein by histidine affinity chromatography as described under “Experimental Procedures.” We started by analyzing the activity of the protein using two different types of salts, KCl and NaCl, using six different concentrations, 5, 10, 25 50, 100, and 200 m m .

    Article Title: Kif2C Minimal Functional Domain Has Unusual Nucleotide Binding Properties That Are Adapted to Microtubule Depolymerization *
    Article Snippet: The HsKif2C cDNA was purchased from Invitrogen, and fragments coding for Kif2C-(N+M) (Ser-187–Gln-598), Kif2C-(sN+M) (Arg-216–Gln-598), and Kif2C-Motor (Asp-252–Gln-598) ( A ) were amplified by PCR and cloned into the NcoI and XhoI pET28a restriction sites for expression. .. All the recombinant Kif2C fragments were expressed in BL21 StarTM (DE3) (Invitrogen) and purified on a HisTrap FF column followed by a Mono S column (GE Healthcare).

    BIA-KA:

    Article Title: A Versatile Strategy for Production of Membrane Proteins with Diverse Topologies: Application to Investigation of Bacterial Homologues of Human Divalent Metal Ion and Nucleoside Transporters
    Article Snippet: Expression trials Expression trials were typically performed in four E . coli strains in parallel: BL21-gold(DE3) (Stratagene), BL21 StarTM (DE3) (Invitrogen; [ ]), C41(DE3) and C43(DE3) (Lucigen Corporation; [ ]), all of which had been transformed with the plasmid pRARE2 (Novagen) to enhance translation efficiency of ORFs with codon usage different from endogenous E . coli genes [ ]. .. Cell lysates were prepared in buffer containing 1% Triton X-100 and 0.1 mg mL-1 lysozyme and their protein concentrations measured using the bicinchoninic acid (BCA) assay (Perbio Science UK Ltd).

    Modification:

    Article Title: A Versatile Strategy for Production of Membrane Proteins with Diverse Topologies: Application to Investigation of Bacterial Homologues of Human Divalent Metal Ion and Nucleoside Transporters
    Article Snippet: Expression trials Expression trials were typically performed in four E . coli strains in parallel: BL21-gold(DE3) (Stratagene), BL21 StarTM (DE3) (Invitrogen; [ ]), C41(DE3) and C43(DE3) (Lucigen Corporation; [ ]), all of which had been transformed with the plasmid pRARE2 (Novagen) to enhance translation efficiency of ORFs with codon usage different from endogenous E . coli genes [ ]. .. Typically, autoinduction of 1 mL cultures in modified M9 medium (M9auto ); Lysogeny broth (LBauto ) or Superbroth medium (SBauto ) containing 0.5% glycerol, 0.05% glucose and 0.2% lactose was performed at 37°C for 24 h in 24-deep-well plates as previously described [ ].

    Western Blot:

    Article Title: A Versatile Strategy for Production of Membrane Proteins with Diverse Topologies: Application to Investigation of Bacterial Homologues of Human Divalent Metal Ion and Nucleoside Transporters
    Article Snippet: Expression trials Expression trials were typically performed in four E . coli strains in parallel: BL21-gold(DE3) (Stratagene), BL21 StarTM (DE3) (Invitrogen; [ ]), C41(DE3) and C43(DE3) (Lucigen Corporation; [ ]), all of which had been transformed with the plasmid pRARE2 (Novagen) to enhance translation efficiency of ORFs with codon usage different from endogenous E . coli genes [ ]. .. Samples were then analysed by SDS-12% (w/v) polyacrylamide gel electrophoresis (SDS-PAGE) followed by staining with Coomassie Brilliant Blue R250 (Fluka) or by western blotting using either horseradish peroxidase-labelled monoclonal antibody against oligohistidine tags or the Strep tag II sequence as appropriate [ ].

    Transformation Assay:

    Article Title: Protein Methionine Sulfoxide Dynamics in Arabidopsis thaliana
    Article Snippet: .. BL21 StarTM One Shot chemically competent Escherichia coli cells (Invitrogen) were transformed, and GSTF9 and GSTT23 protein production was induced with 0.2 m m isopropyl β- d -1-thiogalactopyranoside in growing cultures of successfully transformed E. coli cells reaching an A 600 of 0.8. .. After incubation overnight at 16 °C, cells were centrifuged for 10 min at 2700 × g and resuspended in 20 ml of cold PBS with 1 m m DTT, 1% Triton X-100, and the appropriate amount of Complete protease inhibitor mixture.

    Article Title: Insights into an evolutionary strategy leading to antibiotic resistance
    Article Snippet: The resulting PCR products were cloned into the pET26b(+) vector, and the constructs were then transferred into BL21 StarTM(DE3) (Invitrogen, California, USA). .. Subsequently, the transformed cells were grown on Mueller-Hinton agar plates, supplemented with 50 μg/mL kanamycin and 50–125 μg/mL cefoxitin for selection.

    Article Title: Characterization of Conformation-dependent Prion Protein Epitopes *
    Article Snippet: .. Transformed BL21 StarTM (DE3) (Invitrogen) were cultured until the absorbance value reached 1.0 at 650 nm and induced using isopropyl 1-thio-β- d -galactopyranoside (Sigma) at a final concentration of 1 m m . .. Bacterial pellets were resuspended and lysed with CelLytic solution (Sigma) and centrifuged to obtain inclusion bodies, following solubilization with CelLytic IB solution (Sigma).

    Article Title: Adenine Aminohydrolase from Leishmania donovani
    Article Snippet: .. The pET200/D-TOPO®- AAH construct was then transformed into BL21 StarTM (DE3) One Shot® Escherichia coli (Invitrogen). .. After induction in 0.5 m m isopropyl 1-thio-β- d -galactopyranoside, the recombinant L. donovani AAH protein was purified to virtual homogeneity over a Ni2+ -NTA-agarose column from E. coli extracts that had been prepared using a French press as described previously ( ).

    Article Title: A Versatile Strategy for Production of Membrane Proteins with Diverse Topologies: Application to Investigation of Bacterial Homologues of Human Divalent Metal Ion and Nucleoside Transporters
    Article Snippet: .. Expression trials Expression trials were typically performed in four E . coli strains in parallel: BL21-gold(DE3) (Stratagene), BL21 StarTM (DE3) (Invitrogen; [ ]), C41(DE3) and C43(DE3) (Lucigen Corporation; [ ]), all of which had been transformed with the plasmid pRARE2 (Novagen) to enhance translation efficiency of ORFs with codon usage different from endogenous E . coli genes [ ]. .. Typically, autoinduction of 1 mL cultures in modified M9 medium (M9auto ); Lysogeny broth (LBauto ) or Superbroth medium (SBauto ) containing 0.5% glycerol, 0.05% glucose and 0.2% lactose was performed at 37°C for 24 h in 24-deep-well plates as previously described [ ].

    Protease Inhibitor:

    Article Title: Protein Methionine Sulfoxide Dynamics in Arabidopsis thaliana
    Article Snippet: BL21 StarTM One Shot chemically competent Escherichia coli cells (Invitrogen) were transformed, and GSTF9 and GSTT23 protein production was induced with 0.2 m m isopropyl β- d -1-thiogalactopyranoside in growing cultures of successfully transformed E. coli cells reaching an A 600 of 0.8. .. After incubation overnight at 16 °C, cells were centrifuged for 10 min at 2700 × g and resuspended in 20 ml of cold PBS with 1 m m DTT, 1% Triton X-100, and the appropriate amount of Complete protease inhibitor mixture.

    Cell Culture:

    Article Title: Characterization of Conformation-dependent Prion Protein Epitopes *
    Article Snippet: .. Transformed BL21 StarTM (DE3) (Invitrogen) were cultured until the absorbance value reached 1.0 at 650 nm and induced using isopropyl 1-thio-β- d -galactopyranoside (Sigma) at a final concentration of 1 m m . .. Bacterial pellets were resuspended and lysed with CelLytic solution (Sigma) and centrifuged to obtain inclusion bodies, following solubilization with CelLytic IB solution (Sigma).

    Polymerase Chain Reaction:

    Article Title: A (–)-kolavenyl diphosphate synthase catalyzes the first step of salvinorin A biosynthesis in Salvia divinorum
    Article Snippet: The resulting clones were subsequently transferred to the pEXP-5-CT/TOPO vector by PCR, giving rise to pEXP/Fl-SdKPS and pEXP/Tr-SdKPS vectors, followed by sequence verification. .. The expression of recombinant proteins was carried out in BL21 StarTM (DE3) (ThermoFisher) cells induced via the auto-induction method ( ) at 18 °C to 20 °C.

    Article Title: Insights into an evolutionary strategy leading to antibiotic resistance
    Article Snippet: .. The resulting PCR products were cloned into the pET26b(+) vector, and the constructs were then transferred into BL21 StarTM(DE3) (Invitrogen, California, USA). .. Subsequently, the transformed cells were grown on Mueller-Hinton agar plates, supplemented with 50 μg/mL kanamycin and 50–125 μg/mL cefoxitin for selection.

    Article Title: Characterization of Conformation-dependent Prion Protein Epitopes *
    Article Snippet: Selected shuffled DNA constructs were PCR-amplified and cloned into pET100/D-TOPO® (Invitrogen). .. Transformed BL21 StarTM (DE3) (Invitrogen) were cultured until the absorbance value reached 1.0 at 650 nm and induced using isopropyl 1-thio-β- d -galactopyranoside (Sigma) at a final concentration of 1 m m .

    Article Title: Adenine Aminohydrolase from Leishmania donovani
    Article Snippet: The AAH ORF was amplified by PCR from the AAH cosmid, ligated into the pET200/D-TOPO® bacterial expression vector (Invitrogen), which automatically attaches a His6 tag to the NH2 terminus of the inserted gene product, and sequenced to ensure fidelity of the construct. .. The pET200/D-TOPO®- AAH construct was then transformed into BL21 StarTM (DE3) One Shot® Escherichia coli (Invitrogen).

    Article Title: Kif2C Minimal Functional Domain Has Unusual Nucleotide Binding Properties That Are Adapted to Microtubule Depolymerization *
    Article Snippet: The HsKif2C cDNA was purchased from Invitrogen, and fragments coding for Kif2C-(N+M) (Ser-187–Gln-598), Kif2C-(sN+M) (Arg-216–Gln-598), and Kif2C-Motor (Asp-252–Gln-598) ( A ) were amplified by PCR and cloned into the NcoI and XhoI pET28a restriction sites for expression. .. All the recombinant Kif2C fragments were expressed in BL21 StarTM (DE3) (Invitrogen) and purified on a HisTrap FF column followed by a Mono S column (GE Healthcare).

    Sonication:

    Article Title: Protein Methionine Sulfoxide Dynamics in Arabidopsis thaliana
    Article Snippet: BL21 StarTM One Shot chemically competent Escherichia coli cells (Invitrogen) were transformed, and GSTF9 and GSTT23 protein production was induced with 0.2 m m isopropyl β- d -1-thiogalactopyranoside in growing cultures of successfully transformed E. coli cells reaching an A 600 of 0.8. .. Sonication for 5 min with 10-s pulses was followed by centrifugation for 10 min at 10,000 × g to remove cellular debris after which the recombinant GST proteins were purified from the supernatant by affinity chromatography using immobilized glutathione columns (GSTrap HP, GE Healthcare).

    Article Title: A (–)-kolavenyl diphosphate synthase catalyzes the first step of salvinorin A biosynthesis in Salvia divinorum
    Article Snippet: The expression of recombinant proteins was carried out in BL21 StarTM (DE3) (ThermoFisher) cells induced via the auto-induction method ( ) at 18 °C to 20 °C. .. Pelleted cells were suspended in binding buffer (20 mM HEPES, pH 7.5, 500 mM NaCl, 5 mM imidazole, and 5% glycerol) containing 0.01% (w/v) lysozyme and then disrupted by sonication.

    Article Title: Allergen valency, dose and FcεRI occupancy set thresholds for secretory responses to Pen a 1 and motivate design of hypoallergens
    Article Snippet: All recombinant proteins were expressed in BL21 starTM(DE3) cells (Invitrogen, Grand Island, NY) induced with 1mM isopropylthio-β-galactoside for 4 h. Cells were harvested by centrifugation and stored at −80°C overnight. .. Cells were sonicated using the Misonix system and cell debris was removed by centrifugation at 5000 rpm, 15 min. Proteins were extracted and purified using Ni-NTA purification system (Invitrogen, Grand Island, NY) according to the manufacturer’s instructions using native or denaturing buffer conditions.

    Binding Assay:

    Article Title: A (–)-kolavenyl diphosphate synthase catalyzes the first step of salvinorin A biosynthesis in Salvia divinorum
    Article Snippet: The expression of recombinant proteins was carried out in BL21 StarTM (DE3) (ThermoFisher) cells induced via the auto-induction method ( ) at 18 °C to 20 °C. .. Pelleted cells were suspended in binding buffer (20 mM HEPES, pH 7.5, 500 mM NaCl, 5 mM imidazole, and 5% glycerol) containing 0.01% (w/v) lysozyme and then disrupted by sonication.

    Article Title: Adenine Aminohydrolase from Leishmania donovani
    Article Snippet: The pET200/D-TOPO®- AAH construct was then transformed into BL21 StarTM (DE3) One Shot® Escherichia coli (Invitrogen). .. An additional salt wash containing 32.5 m m imidazole and 1 m sodium chloride was added to reduce nonspecific binding, and the concentration of imidazole in the final wash buffer was increased from 20 to 32.5 m m .

    Mutagenesis:

    Article Title: A (–)-kolavenyl diphosphate synthase catalyzes the first step of salvinorin A biosynthesis in Salvia divinorum
    Article Snippet: Site-directed mutagenesis of Tr-SdKPS was performed using the QuikChange Lightning kit (Agilent) with the pEXP/Tr-SdKPS vector. .. The expression of recombinant proteins was carried out in BL21 StarTM (DE3) (ThermoFisher) cells induced via the auto-induction method ( ) at 18 °C to 20 °C.

    Article Title: Insights into an evolutionary strategy leading to antibiotic resistance
    Article Snippet: For the generation of the library the gene of the target protein was subjected to mutagenesis either via StEP, ePCR or StEP-ePCR , and as implemented by Stevenson and colleagues . .. The resulting PCR products were cloned into the pET26b(+) vector, and the constructs were then transferred into BL21 StarTM(DE3) (Invitrogen, California, USA).

    Article Title: Kif2C Minimal Functional Domain Has Unusual Nucleotide Binding Properties That Are Adapted to Microtubule Depolymerization *
    Article Snippet: G495A mutation was further introduced to abolish the ATP hydrolysis in Kif2C. .. All the recombinant Kif2C fragments were expressed in BL21 StarTM (DE3) (Invitrogen) and purified on a HisTrap FF column followed by a Mono S column (GE Healthcare).

    Size-exclusion Chromatography:

    Article Title: Allergen valency, dose and FcεRI occupancy set thresholds for secretory responses to Pen a 1 and motivate design of hypoallergens
    Article Snippet: All recombinant proteins were expressed in BL21 starTM(DE3) cells (Invitrogen, Grand Island, NY) induced with 1mM isopropylthio-β-galactoside for 4 h. Cells were harvested by centrifugation and stored at −80°C overnight. .. Proteins were dialyzed against phosphate-buffered saline (PBS, pH 7.4) followed by final purification using size exclusion chromatography using the SEC s-3000 column (Phenomenex, Torrance, CA) and the Agilent 1100 system.

    Purification:

    Article Title: Protein Methionine Sulfoxide Dynamics in Arabidopsis thaliana
    Article Snippet: Paragraph title: Recombinant GST Protein Expression and Purification ... BL21 StarTM One Shot chemically competent Escherichia coli cells (Invitrogen) were transformed, and GSTF9 and GSTT23 protein production was induced with 0.2 m m isopropyl β- d -1-thiogalactopyranoside in growing cultures of successfully transformed E. coli cells reaching an A 600 of 0.8.

    Article Title: Characterization of Conformation-dependent Prion Protein Epitopes *
    Article Snippet: Paragraph title: Preparation of Purified Recombinant PrP ... Transformed BL21 StarTM (DE3) (Invitrogen) were cultured until the absorbance value reached 1.0 at 650 nm and induced using isopropyl 1-thio-β- d -galactopyranoside (Sigma) at a final concentration of 1 m m .

    Article Title: Interacting Proteins on Human Spermatozoa: Adaptive Evolution of the Binding of Semenogelin I to EPPIN
    Article Snippet: .. Recombinant proteins were expressed either in BL21 StarTM (DE3) One Shot® E. coli (Invitrogen; SEMG1 constructs) or E. coli Rosetta-Gami 2TM (DE3) (Novagen, Madison, WI; EPPIN construct) and purified under denaturing conditions using nickel-nitrilotriacetic acid agarose (Ni-NTA) beads (Qiagen, Valencia, CA) as described previously [ ]. .. Removal of N-terminal 6X His-tag from recombinant EPPIN was performed using recombinant enterokinase cleavage/capture kit (Novagen), according to the manufacturer’s instructions.

    Article Title: Adenine Aminohydrolase from Leishmania donovani
    Article Snippet: Paragraph title: AAH Expression and Purification ... The pET200/D-TOPO®- AAH construct was then transformed into BL21 StarTM (DE3) One Shot® Escherichia coli (Invitrogen).

    Article Title: Allergen valency, dose and FcεRI occupancy set thresholds for secretory responses to Pen a 1 and motivate design of hypoallergens
    Article Snippet: Paragraph title: Expression and Purification of Recombinant Allergens ... All recombinant proteins were expressed in BL21 starTM(DE3) cells (Invitrogen, Grand Island, NY) induced with 1mM isopropylthio-β-galactoside for 4 h. Cells were harvested by centrifugation and stored at −80°C overnight.

    Article Title: The RNase R from Campylobacter jejuni Has Unique Features and Is Involved in the First Steps of Infection *
    Article Snippet: We tested several conditions for protein expression in different expression strains, and finally, we expressed Cj -RNR at 32 °C in a BL21 StarTM (DE3) (Invitrogen). .. We then purified the protein by histidine affinity chromatography as described under “Experimental Procedures.” We started by analyzing the activity of the protein using two different types of salts, KCl and NaCl, using six different concentrations, 5, 10, 25 50, 100, and 200 m m .

    Article Title: Kif2C Minimal Functional Domain Has Unusual Nucleotide Binding Properties That Are Adapted to Microtubule Depolymerization *
    Article Snippet: .. All the recombinant Kif2C fragments were expressed in BL21 StarTM (DE3) (Invitrogen) and purified on a HisTrap FF column followed by a Mono S column (GE Healthcare). .. More than 10 mg of pure recombinant protein was routinely obtained from a 1-liter Escherichia coli culture.

    Protein Purification:

    Article Title: Kif2C Minimal Functional Domain Has Unusual Nucleotide Binding Properties That Are Adapted to Microtubule Depolymerization *
    Article Snippet: Paragraph title: Constructs and Protein Purification ... All the recombinant Kif2C fragments were expressed in BL21 StarTM (DE3) (Invitrogen) and purified on a HisTrap FF column followed by a Mono S column (GE Healthcare).

    Sequencing:

    Article Title: A (–)-kolavenyl diphosphate synthase catalyzes the first step of salvinorin A biosynthesis in Salvia divinorum
    Article Snippet: The resulting clones were subsequently transferred to the pEXP-5-CT/TOPO vector by PCR, giving rise to pEXP/Fl-SdKPS and pEXP/Tr-SdKPS vectors, followed by sequence verification. .. The expression of recombinant proteins was carried out in BL21 StarTM (DE3) (ThermoFisher) cells induced via the auto-induction method ( ) at 18 °C to 20 °C.

    Article Title: Interacting Proteins on Human Spermatozoa: Adaptive Evolution of the Binding of Semenogelin I to EPPIN
    Article Snippet: Additionally, C-terminal and N-terminal truncated fragments flanking the residue Cys239 from the SEMG1 primary sequence, corresponding to residues R165-R281 (SEMG174-42 ), R165-Q247 (SEMG174-8 ), Q207-R281 (SEMG132-42 ), Y220-L262 (SEMG119-22 ), E229-L269 (SEMG110-30 ), and Q207-Q247 (SEMG132-8 ) ( ) were produced and cloned into the same vector. .. Recombinant proteins were expressed either in BL21 StarTM (DE3) One Shot® E. coli (Invitrogen; SEMG1 constructs) or E. coli Rosetta-Gami 2TM (DE3) (Novagen, Madison, WI; EPPIN construct) and purified under denaturing conditions using nickel-nitrilotriacetic acid agarose (Ni-NTA) beads (Qiagen, Valencia, CA) as described previously [ ].

    Recombinant:

    Article Title: Protein Methionine Sulfoxide Dynamics in Arabidopsis thaliana
    Article Snippet: Paragraph title: Recombinant GST Protein Expression and Purification ... BL21 StarTM One Shot chemically competent Escherichia coli cells (Invitrogen) were transformed, and GSTF9 and GSTT23 protein production was induced with 0.2 m m isopropyl β- d -1-thiogalactopyranoside in growing cultures of successfully transformed E. coli cells reaching an A 600 of 0.8.

    Article Title: A (–)-kolavenyl diphosphate synthase catalyzes the first step of salvinorin A biosynthesis in Salvia divinorum
    Article Snippet: .. The expression of recombinant proteins was carried out in BL21 StarTM (DE3) (ThermoFisher) cells induced via the auto-induction method ( ) at 18 °C to 20 °C. .. Pelleted cells were suspended in binding buffer (20 mM HEPES, pH 7.5, 500 mM NaCl, 5 mM imidazole, and 5% glycerol) containing 0.01% (w/v) lysozyme and then disrupted by sonication.

    Article Title: Characterization of Conformation-dependent Prion Protein Epitopes *
    Article Snippet: Paragraph title: Preparation of Purified Recombinant PrP ... Transformed BL21 StarTM (DE3) (Invitrogen) were cultured until the absorbance value reached 1.0 at 650 nm and induced using isopropyl 1-thio-β- d -galactopyranoside (Sigma) at a final concentration of 1 m m .

    Article Title: Interacting Proteins on Human Spermatozoa: Adaptive Evolution of the Binding of Semenogelin I to EPPIN
    Article Snippet: .. Recombinant proteins were expressed either in BL21 StarTM (DE3) One Shot® E. coli (Invitrogen; SEMG1 constructs) or E. coli Rosetta-Gami 2TM (DE3) (Novagen, Madison, WI; EPPIN construct) and purified under denaturing conditions using nickel-nitrilotriacetic acid agarose (Ni-NTA) beads (Qiagen, Valencia, CA) as described previously [ ]. .. Removal of N-terminal 6X His-tag from recombinant EPPIN was performed using recombinant enterokinase cleavage/capture kit (Novagen), according to the manufacturer’s instructions.

    Article Title: Adenine Aminohydrolase from Leishmania donovani
    Article Snippet: The pET200/D-TOPO®- AAH construct was then transformed into BL21 StarTM (DE3) One Shot® Escherichia coli (Invitrogen). .. After induction in 0.5 m m isopropyl 1-thio-β- d -galactopyranoside, the recombinant L. donovani AAH protein was purified to virtual homogeneity over a Ni2+ -NTA-agarose column from E. coli extracts that had been prepared using a French press as described previously ( ).

    Article Title: Allergen valency, dose and FcεRI occupancy set thresholds for secretory responses to Pen a 1 and motivate design of hypoallergens
    Article Snippet: .. All recombinant proteins were expressed in BL21 starTM(DE3) cells (Invitrogen, Grand Island, NY) induced with 1mM isopropylthio-β-galactoside for 4 h. Cells were harvested by centrifugation and stored at −80°C overnight. ..

    Article Title: Kif2C Minimal Functional Domain Has Unusual Nucleotide Binding Properties That Are Adapted to Microtubule Depolymerization *
    Article Snippet: .. All the recombinant Kif2C fragments were expressed in BL21 StarTM (DE3) (Invitrogen) and purified on a HisTrap FF column followed by a Mono S column (GE Healthcare). .. More than 10 mg of pure recombinant protein was routinely obtained from a 1-liter Escherichia coli culture.

    Affinity Chromatography:

    Article Title: Protein Methionine Sulfoxide Dynamics in Arabidopsis thaliana
    Article Snippet: BL21 StarTM One Shot chemically competent Escherichia coli cells (Invitrogen) were transformed, and GSTF9 and GSTT23 protein production was induced with 0.2 m m isopropyl β- d -1-thiogalactopyranoside in growing cultures of successfully transformed E. coli cells reaching an A 600 of 0.8. .. Sonication for 5 min with 10-s pulses was followed by centrifugation for 10 min at 10,000 × g to remove cellular debris after which the recombinant GST proteins were purified from the supernatant by affinity chromatography using immobilized glutathione columns (GSTrap HP, GE Healthcare).

    Article Title: The RNase R from Campylobacter jejuni Has Unique Features and Is Involved in the First Steps of Infection *
    Article Snippet: We tested several conditions for protein expression in different expression strains, and finally, we expressed Cj -RNR at 32 °C in a BL21 StarTM (DE3) (Invitrogen). .. We then purified the protein by histidine affinity chromatography as described under “Experimental Procedures.” We started by analyzing the activity of the protein using two different types of salts, KCl and NaCl, using six different concentrations, 5, 10, 25 50, 100, and 200 m m .

    Polyacrylamide Gel Electrophoresis:

    Article Title: A Versatile Strategy for Production of Membrane Proteins with Diverse Topologies: Application to Investigation of Bacterial Homologues of Human Divalent Metal Ion and Nucleoside Transporters
    Article Snippet: Expression trials Expression trials were typically performed in four E . coli strains in parallel: BL21-gold(DE3) (Stratagene), BL21 StarTM (DE3) (Invitrogen; [ ]), C41(DE3) and C43(DE3) (Lucigen Corporation; [ ]), all of which had been transformed with the plasmid pRARE2 (Novagen) to enhance translation efficiency of ORFs with codon usage different from endogenous E . coli genes [ ]. .. Samples were then analysed by SDS-12% (w/v) polyacrylamide gel electrophoresis (SDS-PAGE) followed by staining with Coomassie Brilliant Blue R250 (Fluka) or by western blotting using either horseradish peroxidase-labelled monoclonal antibody against oligohistidine tags or the Strep tag II sequence as appropriate [ ].

    Chloramphenicol Acetyltransferase Assay:

    Article Title: Insights into an evolutionary strategy leading to antibiotic resistance
    Article Snippet: The forward and reverse primer sequences were 5′-GGA GAT ATA CAT ATG AAA CGT CGC TTC ACC-3′ and 5′-AAG CTT GAA TTC TTA CGG GCG AGC ACC GCT-3, respectively. .. The resulting PCR products were cloned into the pET26b(+) vector, and the constructs were then transferred into BL21 StarTM(DE3) (Invitrogen, California, USA).

    SDS Page:

    Article Title: A Versatile Strategy for Production of Membrane Proteins with Diverse Topologies: Application to Investigation of Bacterial Homologues of Human Divalent Metal Ion and Nucleoside Transporters
    Article Snippet: Expression trials Expression trials were typically performed in four E . coli strains in parallel: BL21-gold(DE3) (Stratagene), BL21 StarTM (DE3) (Invitrogen; [ ]), C41(DE3) and C43(DE3) (Lucigen Corporation; [ ]), all of which had been transformed with the plasmid pRARE2 (Novagen) to enhance translation efficiency of ORFs with codon usage different from endogenous E . coli genes [ ]. .. Samples were then analysed by SDS-12% (w/v) polyacrylamide gel electrophoresis (SDS-PAGE) followed by staining with Coomassie Brilliant Blue R250 (Fluka) or by western blotting using either horseradish peroxidase-labelled monoclonal antibody against oligohistidine tags or the Strep tag II sequence as appropriate [ ].

    Plasmid Preparation:

    Article Title: A (–)-kolavenyl diphosphate synthase catalyzes the first step of salvinorin A biosynthesis in Salvia divinorum
    Article Snippet: SdKSL1 and SdKSL3 were subcloned into the pET15b vector (Merck), and SdKSL2 was subcloned into the pCOLADuetTM -1 (Novagen) expression plasmid. .. The expression of recombinant proteins was carried out in BL21 StarTM (DE3) (ThermoFisher) cells induced via the auto-induction method ( ) at 18 °C to 20 °C.

    Article Title: Insights into an evolutionary strategy leading to antibiotic resistance
    Article Snippet: .. The resulting PCR products were cloned into the pET26b(+) vector, and the constructs were then transferred into BL21 StarTM(DE3) (Invitrogen, California, USA). .. Subsequently, the transformed cells were grown on Mueller-Hinton agar plates, supplemented with 50 μg/mL kanamycin and 50–125 μg/mL cefoxitin for selection.

    Article Title: Interacting Proteins on Human Spermatozoa: Adaptive Evolution of the Binding of Semenogelin I to EPPIN
    Article Snippet: Additionally, C-terminal and N-terminal truncated fragments flanking the residue Cys239 from the SEMG1 primary sequence, corresponding to residues R165-R281 (SEMG174-42 ), R165-Q247 (SEMG174-8 ), Q207-R281 (SEMG132-42 ), Y220-L262 (SEMG119-22 ), E229-L269 (SEMG110-30 ), and Q207-Q247 (SEMG132-8 ) ( ) were produced and cloned into the same vector. .. Recombinant proteins were expressed either in BL21 StarTM (DE3) One Shot® E. coli (Invitrogen; SEMG1 constructs) or E. coli Rosetta-Gami 2TM (DE3) (Novagen, Madison, WI; EPPIN construct) and purified under denaturing conditions using nickel-nitrilotriacetic acid agarose (Ni-NTA) beads (Qiagen, Valencia, CA) as described previously [ ].

    Article Title: Adenine Aminohydrolase from Leishmania donovani
    Article Snippet: The AAH ORF was amplified by PCR from the AAH cosmid, ligated into the pET200/D-TOPO® bacterial expression vector (Invitrogen), which automatically attaches a His6 tag to the NH2 terminus of the inserted gene product, and sequenced to ensure fidelity of the construct. .. The pET200/D-TOPO®- AAH construct was then transformed into BL21 StarTM (DE3) One Shot® Escherichia coli (Invitrogen).

    Article Title: A Versatile Strategy for Production of Membrane Proteins with Diverse Topologies: Application to Investigation of Bacterial Homologues of Human Divalent Metal Ion and Nucleoside Transporters
    Article Snippet: .. Expression trials Expression trials were typically performed in four E . coli strains in parallel: BL21-gold(DE3) (Stratagene), BL21 StarTM (DE3) (Invitrogen; [ ]), C41(DE3) and C43(DE3) (Lucigen Corporation; [ ]), all of which had been transformed with the plasmid pRARE2 (Novagen) to enhance translation efficiency of ORFs with codon usage different from endogenous E . coli genes [ ]. .. Typically, autoinduction of 1 mL cultures in modified M9 medium (M9auto ); Lysogeny broth (LBauto ) or Superbroth medium (SBauto ) containing 0.5% glycerol, 0.05% glucose and 0.2% lactose was performed at 37°C for 24 h in 24-deep-well plates as previously described [ ].

    Article Title: The RNase R from Campylobacter jejuni Has Unique Features and Is Involved in the First Steps of Infection *
    Article Snippet: We have cloned the rnr gene from C. jejuni in an expression vector. .. We tested several conditions for protein expression in different expression strains, and finally, we expressed Cj -RNR at 32 °C in a BL21 StarTM (DE3) (Invitrogen).

    Selection:

    Article Title: Insights into an evolutionary strategy leading to antibiotic resistance
    Article Snippet: Directed evolution Directed evolution involved two major steps, i.e . library generation and selection. .. The resulting PCR products were cloned into the pET26b(+) vector, and the constructs were then transferred into BL21 StarTM(DE3) (Invitrogen, California, USA).

    Produced:

    Article Title: Interacting Proteins on Human Spermatozoa: Adaptive Evolution of the Binding of Semenogelin I to EPPIN
    Article Snippet: Additionally, C-terminal and N-terminal truncated fragments flanking the residue Cys239 from the SEMG1 primary sequence, corresponding to residues R165-R281 (SEMG174-42 ), R165-Q247 (SEMG174-8 ), Q207-R281 (SEMG132-42 ), Y220-L262 (SEMG119-22 ), E229-L269 (SEMG110-30 ), and Q207-Q247 (SEMG132-8 ) ( ) were produced and cloned into the same vector. .. Recombinant proteins were expressed either in BL21 StarTM (DE3) One Shot® E. coli (Invitrogen; SEMG1 constructs) or E. coli Rosetta-Gami 2TM (DE3) (Novagen, Madison, WI; EPPIN construct) and purified under denaturing conditions using nickel-nitrilotriacetic acid agarose (Ni-NTA) beads (Qiagen, Valencia, CA) as described previously [ ].

    Concentration Assay:

    Article Title: Insights into an evolutionary strategy leading to antibiotic resistance
    Article Snippet: The resulting PCR products were cloned into the pET26b(+) vector, and the constructs were then transferred into BL21 StarTM(DE3) (Invitrogen, California, USA). .. To ensure a low expression level a final concentration of 50 μM isopropyl-β-D-1- thiogalactopyranoside (IPTG) (Solon, OH, USA) was used in all selection media.

    Article Title: Characterization of Conformation-dependent Prion Protein Epitopes *
    Article Snippet: .. Transformed BL21 StarTM (DE3) (Invitrogen) were cultured until the absorbance value reached 1.0 at 650 nm and induced using isopropyl 1-thio-β- d -galactopyranoside (Sigma) at a final concentration of 1 m m . .. Bacterial pellets were resuspended and lysed with CelLytic solution (Sigma) and centrifuged to obtain inclusion bodies, following solubilization with CelLytic IB solution (Sigma).

    Article Title: Adenine Aminohydrolase from Leishmania donovani
    Article Snippet: The pET200/D-TOPO®- AAH construct was then transformed into BL21 StarTM (DE3) One Shot® Escherichia coli (Invitrogen). .. An additional salt wash containing 32.5 m m imidazole and 1 m sodium chloride was added to reduce nonspecific binding, and the concentration of imidazole in the final wash buffer was increased from 20 to 32.5 m m .

    Strep-tag:

    Article Title: A Versatile Strategy for Production of Membrane Proteins with Diverse Topologies: Application to Investigation of Bacterial Homologues of Human Divalent Metal Ion and Nucleoside Transporters
    Article Snippet: Expression trials Expression trials were typically performed in four E . coli strains in parallel: BL21-gold(DE3) (Stratagene), BL21 StarTM (DE3) (Invitrogen; [ ]), C41(DE3) and C43(DE3) (Lucigen Corporation; [ ]), all of which had been transformed with the plasmid pRARE2 (Novagen) to enhance translation efficiency of ORFs with codon usage different from endogenous E . coli genes [ ]. .. Samples were then analysed by SDS-12% (w/v) polyacrylamide gel electrophoresis (SDS-PAGE) followed by staining with Coomassie Brilliant Blue R250 (Fluka) or by western blotting using either horseradish peroxidase-labelled monoclonal antibody against oligohistidine tags or the Strep tag II sequence as appropriate [ ].

    Staining:

    Article Title: A Versatile Strategy for Production of Membrane Proteins with Diverse Topologies: Application to Investigation of Bacterial Homologues of Human Divalent Metal Ion and Nucleoside Transporters
    Article Snippet: Expression trials Expression trials were typically performed in four E . coli strains in parallel: BL21-gold(DE3) (Stratagene), BL21 StarTM (DE3) (Invitrogen; [ ]), C41(DE3) and C43(DE3) (Lucigen Corporation; [ ]), all of which had been transformed with the plasmid pRARE2 (Novagen) to enhance translation efficiency of ORFs with codon usage different from endogenous E . coli genes [ ]. .. Samples were then analysed by SDS-12% (w/v) polyacrylamide gel electrophoresis (SDS-PAGE) followed by staining with Coomassie Brilliant Blue R250 (Fluka) or by western blotting using either horseradish peroxidase-labelled monoclonal antibody against oligohistidine tags or the Strep tag II sequence as appropriate [ ].

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