bl21  (New England Biolabs)


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    Structured Review

    New England Biolabs bl21
    Aggregation of disulfide-reduced SOD1 with fALS mutations in E. coli <t>BL21</t> was not rescued by addition of ZnSO 4 . E. coli BL21(DE3) was transformed with pET15b harboring human SOD1 cDNA with indicated fALS mutations, and the protein expression was induced by IPTG in the presence of 1 mM ZnSO 4 . Cell lysates were fractionated into soluble supernatant (s) and insoluble pellets (p), treated with iodoacetamide, and then analyzed with non-reducing SDS-PAGE by using a 15% polyacrylamide gel. White (SOD1 S-S ) and black (SOD1 SH ) arrows at the right side of the gel image indicate positions of bands corresponding to SOD1 with and without a disulfide bond, respectively.
    Bl21, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/bl21/product/New England Biolabs
    Average 97 stars, based on 1 article reviews
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    bl21 - by Bioz Stars, 2022-05
    97/100 stars

    Images

    1) Product Images from "Redox environment is an intracellular factor to operate distinct pathways for aggregation of Cu,Zn-superoxide dismutase in amyotrophic lateral sclerosis"

    Article Title: Redox environment is an intracellular factor to operate distinct pathways for aggregation of Cu,Zn-superoxide dismutase in amyotrophic lateral sclerosis

    Journal: Frontiers in Cellular Neuroscience

    doi: 10.3389/fncel.2013.00240

    Aggregation of disulfide-reduced SOD1 with fALS mutations in E. coli BL21 was not rescued by addition of ZnSO 4 . E. coli BL21(DE3) was transformed with pET15b harboring human SOD1 cDNA with indicated fALS mutations, and the protein expression was induced by IPTG in the presence of 1 mM ZnSO 4 . Cell lysates were fractionated into soluble supernatant (s) and insoluble pellets (p), treated with iodoacetamide, and then analyzed with non-reducing SDS-PAGE by using a 15% polyacrylamide gel. White (SOD1 S-S ) and black (SOD1 SH ) arrows at the right side of the gel image indicate positions of bands corresponding to SOD1 with and without a disulfide bond, respectively.
    Figure Legend Snippet: Aggregation of disulfide-reduced SOD1 with fALS mutations in E. coli BL21 was not rescued by addition of ZnSO 4 . E. coli BL21(DE3) was transformed with pET15b harboring human SOD1 cDNA with indicated fALS mutations, and the protein expression was induced by IPTG in the presence of 1 mM ZnSO 4 . Cell lysates were fractionated into soluble supernatant (s) and insoluble pellets (p), treated with iodoacetamide, and then analyzed with non-reducing SDS-PAGE by using a 15% polyacrylamide gel. White (SOD1 S-S ) and black (SOD1 SH ) arrows at the right side of the gel image indicate positions of bands corresponding to SOD1 with and without a disulfide bond, respectively.

    Techniques Used: Transformation Assay, Expressing, SDS Page

    Amyloid-like characters of SOD1 aggregates purified from insoluble inclusions in E. coli . (A) SOD1(G37R) aggregates purified from insoluble inclusions in E. coli were reacted with iodoacetamide for protection of free thiol groups and analyzed with reducing (+β-ME) and non-reducing (+IA) SDS-PAGE. Disulfide-crosslinked oligomers were identified in SOD1(G37R) aggregates purified from insoluble inclusions in E. coli SHuffle TM but not in BL21(DE3). (B, C) Tinctorial properties of SOD1(G37R) aggregates purified from E. coli BL21 and SHuffle TM (red and blue curves, respectively) were examined by (B) fluorescence of thioflavin T and (C) absorption of Congo red. Black curves represent (B) fluorescence spectrum of thioflavin T and (C) absorption spectrum of Congo red without addition of SOD1 aggregates. (D, E) Electron micrograms of SOD1(G37R) aggregates purified from E. coli (D) BL21 and (E) SHuffle TM . SOD1(G37R) aggregates exhibit large, amorphous morphologies (left panels), while fibrillar structures become evident after brief treatment of aggregates with Proteinase K (right panels). A bar represents 2 μm (left panels) or 50 nm (right panels).
    Figure Legend Snippet: Amyloid-like characters of SOD1 aggregates purified from insoluble inclusions in E. coli . (A) SOD1(G37R) aggregates purified from insoluble inclusions in E. coli were reacted with iodoacetamide for protection of free thiol groups and analyzed with reducing (+β-ME) and non-reducing (+IA) SDS-PAGE. Disulfide-crosslinked oligomers were identified in SOD1(G37R) aggregates purified from insoluble inclusions in E. coli SHuffle TM but not in BL21(DE3). (B, C) Tinctorial properties of SOD1(G37R) aggregates purified from E. coli BL21 and SHuffle TM (red and blue curves, respectively) were examined by (B) fluorescence of thioflavin T and (C) absorption of Congo red. Black curves represent (B) fluorescence spectrum of thioflavin T and (C) absorption spectrum of Congo red without addition of SOD1 aggregates. (D, E) Electron micrograms of SOD1(G37R) aggregates purified from E. coli (D) BL21 and (E) SHuffle TM . SOD1(G37R) aggregates exhibit large, amorphous morphologies (left panels), while fibrillar structures become evident after brief treatment of aggregates with Proteinase K (right panels). A bar represents 2 μm (left panels) or 50 nm (right panels).

    Techniques Used: Purification, IA, SDS Page, Fluorescence

    2) Product Images from "Safe Recombinant Outer Membrane Vesicles that Display M2e Elicit Heterologous Influenza Protection"

    Article Title: Safe Recombinant Outer Membrane Vesicles that Display M2e Elicit Heterologous Influenza Protection

    Journal: Molecular Therapy

    doi: 10.1016/j.ymthe.2017.01.010

    Pyrogenicity and TLR Activity Are rOMV Source Strain Dependent (A) Pyrogenicity (measured in endotoxin units) of CC, Nsl, and BL21 rOMVS determined using whole-blood pyrogenicity test. Groups were compared with Kruskal-Wallis test, followed by Mann-Whitney between pairs, using Bonferroni method to account for multiple comparisons (*p
    Figure Legend Snippet: Pyrogenicity and TLR Activity Are rOMV Source Strain Dependent (A) Pyrogenicity (measured in endotoxin units) of CC, Nsl, and BL21 rOMVS determined using whole-blood pyrogenicity test. Groups were compared with Kruskal-Wallis test, followed by Mann-Whitney between pairs, using Bonferroni method to account for multiple comparisons (*p

    Techniques Used: Activity Assay, MANN-WHITNEY

    rOMVs Produced from Three Different E. coli Strains Are Structurally Comparable (A–C) TEM images of rOMVs stained with uranyl acetate: CC rOMVs (A), BL21 rOMVs (B), and Nsl rOMVs (C). Scale bars represent 100 nm. (D and E) Total IgG (D) and isotypes IgG1 and IgG2a (E) anti-GFP titers from BALB/c mice 8 weeks post prime dose of ClyA-GFP-expressing CC or BL21 rOMVs. Titer error bars represent 95% confidence intervals (CI) of geometric mean. Log-transformed data analyzed using an unpaired Student’s t test to compare CC versus BL21 rOMV anti-GFP IgG levels and using paired Student’s t test to compare IgG1:IgG2a levels for each rOMV type. Dotted line indicates titer of sera from mice pre-vaccination.
    Figure Legend Snippet: rOMVs Produced from Three Different E. coli Strains Are Structurally Comparable (A–C) TEM images of rOMVs stained with uranyl acetate: CC rOMVs (A), BL21 rOMVs (B), and Nsl rOMVs (C). Scale bars represent 100 nm. (D and E) Total IgG (D) and isotypes IgG1 and IgG2a (E) anti-GFP titers from BALB/c mice 8 weeks post prime dose of ClyA-GFP-expressing CC or BL21 rOMVs. Titer error bars represent 95% confidence intervals (CI) of geometric mean. Log-transformed data analyzed using an unpaired Student’s t test to compare CC versus BL21 rOMV anti-GFP IgG levels and using paired Student’s t test to compare IgG1:IgG2a levels for each rOMV type. Dotted line indicates titer of sera from mice pre-vaccination.

    Techniques Used: Produced, Transmission Electron Microscopy, Staining, Mouse Assay, Expressing, Transformation Assay

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    New England Biolabs e coli bl21 strains
    Functional validation of the predicted ARGs and structural investigation of conserved sites detected by HMD-ARG. a Left figure: A diagram showing the procedure of heterologous expression and functional analysis of the predicted ARGs from PA150567 in E. coli <t>BL21</t> host. Middle figure : Growth curves of E. coli hosts with expression of the predicted β -lactamases that inactivate β -lactam antibiotics in the presence of 2 μg/ml meropenem. Right figure : Growth curves of E. coli hosts with expression of the predicted acetyltransferases that inactivate aminoglycoside antibiotics in the presence of 4 μg/ml amikacin. b The HMD-ARG predicted conserved sites and the corresponding sequence logo from 319 to 393 in AXX01_04100. c After we mutated the conserved site (346) from T to N, the mutated protein’s (colored in red) local structure (predicted by RaptorX) changed significantly from the wild type (colored in light blue). Moreover, the binding affinity (predicted by AutoDock) between the mutated protein and the ligand (antibiotics) also reduced, as illustrated in the middle (wild type) and right (mutated protein) figures
    E Coli Bl21 Strains, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Functional validation of the predicted ARGs and structural investigation of conserved sites detected by HMD-ARG. a Left figure: A diagram showing the procedure of heterologous expression and functional analysis of the predicted ARGs from PA150567 in E. coli BL21 host. Middle figure : Growth curves of E. coli hosts with expression of the predicted β -lactamases that inactivate β -lactam antibiotics in the presence of 2 μg/ml meropenem. Right figure : Growth curves of E. coli hosts with expression of the predicted acetyltransferases that inactivate aminoglycoside antibiotics in the presence of 4 μg/ml amikacin. b The HMD-ARG predicted conserved sites and the corresponding sequence logo from 319 to 393 in AXX01_04100. c After we mutated the conserved site (346) from T to N, the mutated protein’s (colored in red) local structure (predicted by RaptorX) changed significantly from the wild type (colored in light blue). Moreover, the binding affinity (predicted by AutoDock) between the mutated protein and the ligand (antibiotics) also reduced, as illustrated in the middle (wild type) and right (mutated protein) figures

    Journal: Microbiome

    Article Title: HMD-ARG: hierarchical multi-task deep learning for annotating antibiotic resistance genes

    doi: 10.1186/s40168-021-01002-3

    Figure Lengend Snippet: Functional validation of the predicted ARGs and structural investigation of conserved sites detected by HMD-ARG. a Left figure: A diagram showing the procedure of heterologous expression and functional analysis of the predicted ARGs from PA150567 in E. coli BL21 host. Middle figure : Growth curves of E. coli hosts with expression of the predicted β -lactamases that inactivate β -lactam antibiotics in the presence of 2 μg/ml meropenem. Right figure : Growth curves of E. coli hosts with expression of the predicted acetyltransferases that inactivate aminoglycoside antibiotics in the presence of 4 μg/ml amikacin. b The HMD-ARG predicted conserved sites and the corresponding sequence logo from 319 to 393 in AXX01_04100. c After we mutated the conserved site (346) from T to N, the mutated protein’s (colored in red) local structure (predicted by RaptorX) changed significantly from the wild type (colored in light blue). Moreover, the binding affinity (predicted by AutoDock) between the mutated protein and the ligand (antibiotics) also reduced, as illustrated in the middle (wild type) and right (mutated protein) figures

    Article Snippet: Overnight culture of E. coli BL21 strains containing the plasmids for overexpression of the predicted genes were 1:100 diluted and grown in LB medium supplemented with kanamycin (20 μg/ml) and Isopropyl β-D-1-thiogalactopyranoside (IPTG) (0.5 mM).

    Techniques: Functional Assay, Expressing, Sequencing, Binding Assay

    Use of THP-1 TLR4-CD14 NF-κB-eGFP reporter cells for the detection of microbial contaminations in recombinant protein preparations. (A) THP-1 TLR4-CD14 NF-κB-eGFP reporter cells were incubated with the indicated serial dilutions of ultrapure LPS for 24 h. NF-κB-driven eGFP expression was assessed by flow cytometry. Bar graphs show geometric mean of fluorescence intensity (gMFI). eGFP expression of the parental THP-1 reporter cells treated with 300 ng/ml ultrapure LPS is shown for comparison (grey bar). (B) mRNA expression of TLR4, CD14 and MD2 was measured in parental and TLR4-CD14 THP-1 reporter cells by real-time qPCR. Data are presented as mean ± SEM of expression values normalized against the housekeeping gene GAPDH (n = 4). (C) Recombinant human split product C4dg produced in three different expression systems (HEK293-6E, standard E . coli BL21 and E . coli ClearColi BL21) was tested for TLR4-agonist contaminations using HEK293 hTLR4A-MD2-CD14 cells. Following 24 h of incubation the IL-8 content in the culture supernatants was measured by ELISA. (D) Parental and TLR4-CD14 THP-1 reporters were incubated with the protein preparations described in (C) and NF-κB-driven eGFP expression was assessed by flow cytometry 24 h later. Bar graphs show geometric mean of fluorescence intensity (gMFI). Mean and SE were calculated from duplicates of four independently performed experiments (n = 4) (E) Immature human moDCs were incubated with standard LPS, E . coli BL21 or E . coli ClearColi BL21 expressed C4dg protein at the indicated concentrations for 24 h or were left untreated. Expression of maturation markers CD83 and CD86 was assessed by flow cytometry. (F) E . coli ClearColi BL21-expressed C4dg protein was subjected to a single round of bulk chromatography using a polymyxin resin (see Material and Methods ). Samples before and after chromatography were tested using the THP-1 TLR4-CD14 reporters. Mean and SE were calculated from duplicates of four independently performed experiments (n = 4).

    Journal: PLoS ONE

    Article Title: A human monocytic NF-κB fluorescent reporter cell line for detection of microbial contaminants in biological samples

    doi: 10.1371/journal.pone.0178220

    Figure Lengend Snippet: Use of THP-1 TLR4-CD14 NF-κB-eGFP reporter cells for the detection of microbial contaminations in recombinant protein preparations. (A) THP-1 TLR4-CD14 NF-κB-eGFP reporter cells were incubated with the indicated serial dilutions of ultrapure LPS for 24 h. NF-κB-driven eGFP expression was assessed by flow cytometry. Bar graphs show geometric mean of fluorescence intensity (gMFI). eGFP expression of the parental THP-1 reporter cells treated with 300 ng/ml ultrapure LPS is shown for comparison (grey bar). (B) mRNA expression of TLR4, CD14 and MD2 was measured in parental and TLR4-CD14 THP-1 reporter cells by real-time qPCR. Data are presented as mean ± SEM of expression values normalized against the housekeeping gene GAPDH (n = 4). (C) Recombinant human split product C4dg produced in three different expression systems (HEK293-6E, standard E . coli BL21 and E . coli ClearColi BL21) was tested for TLR4-agonist contaminations using HEK293 hTLR4A-MD2-CD14 cells. Following 24 h of incubation the IL-8 content in the culture supernatants was measured by ELISA. (D) Parental and TLR4-CD14 THP-1 reporters were incubated with the protein preparations described in (C) and NF-κB-driven eGFP expression was assessed by flow cytometry 24 h later. Bar graphs show geometric mean of fluorescence intensity (gMFI). Mean and SE were calculated from duplicates of four independently performed experiments (n = 4) (E) Immature human moDCs were incubated with standard LPS, E . coli BL21 or E . coli ClearColi BL21 expressed C4dg protein at the indicated concentrations for 24 h or were left untreated. Expression of maturation markers CD83 and CD86 was assessed by flow cytometry. (F) E . coli ClearColi BL21-expressed C4dg protein was subjected to a single round of bulk chromatography using a polymyxin resin (see Material and Methods ). Samples before and after chromatography were tested using the THP-1 TLR4-CD14 reporters. Mean and SE were calculated from duplicates of four independently performed experiments (n = 4).

    Article Snippet: The electrocompetent E. coli protein expression strain ClearColi BL21 was purchased from Lucigen (Middleton, WI) and standard E. coli BL21 were obtained from New England Biolabs (Ipswich, MA).

    Techniques: Recombinant, Incubation, Expressing, Flow Cytometry, Cytometry, Fluorescence, Real-time Polymerase Chain Reaction, Produced, Enzyme-linked Immunosorbent Assay, Chromatography

    pTF.CREG1 expression and purification. Briefly, genes encoding various pTF.CREG1 truncations were placed under arabinose-inducible promoter, expressed in BL21 E. coli cells, and purified using cobalt, Co 2+ , resin-based gravity flow columns. CTHF: C-terminal His6 and FLAG tag. NTH: N-terminal His6 tag. L: PageRuler Plus Prestained Protein Ladder. (A) Final elutions of various pTF.CREG1 truncations from small scale (50 mL) protein expression experiments. Δ31_pTF.CREG1-His6 (bolded and black rectangle) was selected for scale-up. (B) The final Δ31_pTF.CREG1-His6 elution from a large-scale (2 L) protein expression experiment. NuPage 4-12% Bis-Tris gels were used to resolve protein samples.

    Journal: bioRxiv

    Article Title: Proximity proteomics in a marine diatom reveals a putative cell surface-to-chloroplast iron trafficking pathway

    doi: 10.1101/806539

    Figure Lengend Snippet: pTF.CREG1 expression and purification. Briefly, genes encoding various pTF.CREG1 truncations were placed under arabinose-inducible promoter, expressed in BL21 E. coli cells, and purified using cobalt, Co 2+ , resin-based gravity flow columns. CTHF: C-terminal His6 and FLAG tag. NTH: N-terminal His6 tag. L: PageRuler Plus Prestained Protein Ladder. (A) Final elutions of various pTF.CREG1 truncations from small scale (50 mL) protein expression experiments. Δ31_pTF.CREG1-His6 (bolded and black rectangle) was selected for scale-up. (B) The final Δ31_pTF.CREG1-His6 elution from a large-scale (2 L) protein expression experiment. NuPage 4-12% Bis-Tris gels were used to resolve protein samples.

    Article Snippet: Assembly, colony PCR screening, plasmid isolation, sequencing, and transformation into chemically competent BL21 E. coli cells of all additional expression vectors, including PtpBAD-Δ31_pTF.CREG1-CTHF (=pJT_Δ31_pTF.CREG1-His6), was performed as above.

    Techniques: Expressing, Purification, FLAG-tag

    Sender plasmids expressed in Escherichia coli BL21. (A) The modular Sender vector allows facile cloning of any EcoRI , XbaI ] glyphs (top) and a scaled circular map (bottom). (B) Optical density (OD) readings for E . coli cultures expressing each of the Sender plasmids. (C) mCherry expression is shown as end-point RFP signal normalized to OD 600 after an 8 hour growth period.

    Journal: PLoS ONE

    Article Title: Characterization of diverse homoserine lactone synthases in Escherichia coli

    doi: 10.1371/journal.pone.0202294

    Figure Lengend Snippet: Sender plasmids expressed in Escherichia coli BL21. (A) The modular Sender vector allows facile cloning of any EcoRI , XbaI ] glyphs (top) and a scaled circular map (bottom). (B) Optical density (OD) readings for E . coli cultures expressing each of the Sender plasmids. (C) mCherry expression is shown as end-point RFP signal normalized to OD 600 after an 8 hour growth period.

    Article Snippet: Cultures of E . coli BL21 (New England Biolabs) transformed with Sender plasmids, a Receiver plasmid, a negative receiver plasmid, and a GFP positive plasmid were grown in 3 mL of LB with 5 ug/mL ampicillin for 16 hours at 37°C and shaking at 220 rpm.

    Techniques: Plasmid Preparation, Clone Assay, Expressing