bl21  (New England Biolabs)


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    BL21 Competent E coli
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    BL21 Competent E coli 20x0 05 ml
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    c2530h
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    Competent Bacteria
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    New England Biolabs bl21
    BL21 Competent E coli
    BL21 Competent E coli 20x0 05 ml
    https://www.bioz.com/result/bl21/product/New England Biolabs
    Average 99 stars, based on 216 article reviews
    Price from $9.99 to $1999.99
    bl21 - by Bioz Stars, 2020-08
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    Images

    1) Product Images from "A Cyan Fluorescent Reporter Expressed from the Chloroplast Genome of Marchantia polymorpha"

    Article Title: A Cyan Fluorescent Reporter Expressed from the Chloroplast Genome of Marchantia polymorpha

    Journal: Plant and Cell Physiology

    doi: 10.1093/pcp/pcv160

    Detection of chloroplast-expressed mTurquoise2 in transgenic lines of Marchantia polymorpha. (A) M. polymorpha protein extracts separated by SDS–PAGE. Protein extracts from wild type (WT) and transplastomic (CL0*b) lines of M. polymorpha were separated by 4–12% SDS–PAGE, and visualized by in-gel fluorescence (left) and subsequent Coomassie stain (right). The fluorescence image was generated using a custom imaging device described in the Materials and Methods for visualization of CFP bands (emission 486/10 nm, green) and marker (emission 540/10 nm, red). (B) Untagged and His 6 -tagged mTurquoise2 separated by SDS–PAGE. Untagged mTurquoise2 was expressed from plasmids pCS CL0*b ( psbA promoter) in BL21 E. coli and pCRB SREI (T7 promoter) in T7 Express E. coli. His 6 -tagged mTurquoise2 was expressed from plasmid pCRB SREI 6his (T7 promoter) and purified by affinity chromatography. The rightmost lanes contain 50 ng of unboiled and boiled purified protein, respectively. Visualization by in-gel fluorescence and Coomassie stain was conducted as described above. (C) Standard curve for quantification of mTurquoise2 based on in-gel fluorescence. Serial dilutions of 1 µg purified of mTurquoise2 were separated by 4–12% SDS–PAGE and the band intensity quantified by in-gel fluorescence as described in the Materials and Methods. Levels of mTurquoise2 extracted from transplastomic (CL0*b) M. polymorpha (red cross) were estimated by linear regression analysis on densities of fluorescent target bands as shown in (A). Error bars represent the SD of average band density between three different experiments.
    Figure Legend Snippet: Detection of chloroplast-expressed mTurquoise2 in transgenic lines of Marchantia polymorpha. (A) M. polymorpha protein extracts separated by SDS–PAGE. Protein extracts from wild type (WT) and transplastomic (CL0*b) lines of M. polymorpha were separated by 4–12% SDS–PAGE, and visualized by in-gel fluorescence (left) and subsequent Coomassie stain (right). The fluorescence image was generated using a custom imaging device described in the Materials and Methods for visualization of CFP bands (emission 486/10 nm, green) and marker (emission 540/10 nm, red). (B) Untagged and His 6 -tagged mTurquoise2 separated by SDS–PAGE. Untagged mTurquoise2 was expressed from plasmids pCS CL0*b ( psbA promoter) in BL21 E. coli and pCRB SREI (T7 promoter) in T7 Express E. coli. His 6 -tagged mTurquoise2 was expressed from plasmid pCRB SREI 6his (T7 promoter) and purified by affinity chromatography. The rightmost lanes contain 50 ng of unboiled and boiled purified protein, respectively. Visualization by in-gel fluorescence and Coomassie stain was conducted as described above. (C) Standard curve for quantification of mTurquoise2 based on in-gel fluorescence. Serial dilutions of 1 µg purified of mTurquoise2 were separated by 4–12% SDS–PAGE and the band intensity quantified by in-gel fluorescence as described in the Materials and Methods. Levels of mTurquoise2 extracted from transplastomic (CL0*b) M. polymorpha (red cross) were estimated by linear regression analysis on densities of fluorescent target bands as shown in (A). Error bars represent the SD of average band density between three different experiments.

    Techniques Used: Transgenic Assay, SDS Page, Fluorescence, Staining, Generated, Imaging, Marker, Plasmid Preparation, Purification, Affinity Chromatography

    2) Product Images from "Improve Protein Solubility and Activity based on Machine Learning Models"

    Article Title: Improve Protein Solubility and Activity based on Machine Learning Models

    Journal: bioRxiv

    doi: 10.1101/817890

    ( a) The SDS-PAGE analysis of protein tal and dxs expressed in E. coli with and without tags designed by our optimization algorithm. “+” and “-” represented expressed proteins with and without peptide tags respectively. “P” and “S” represented the pellet fraction (insoluble) and supernatant fraction (soluble), respectively. The oval shapes highlight the bands of dxs and tal proteins. Protein tal and dxs were expressed in K3 medium with 20 g/L glucose at 30 °C. ( b) Quantitative presentation of the SDS-PAGE images in a . The protein solubility was the ratio of soluble protein amount to the total protein amount. The protein amount was estimated by using band intensity on SDS-PAGE images. The sequences of the designed tags for N-terminal and C-terminal were shown. The amino acid S and G on the two ends of the tags were the linkers for GT DNA assembly standard, which was used to construct the plasmids in this study 40 .
    Figure Legend Snippet: ( a) The SDS-PAGE analysis of protein tal and dxs expressed in E. coli with and without tags designed by our optimization algorithm. “+” and “-” represented expressed proteins with and without peptide tags respectively. “P” and “S” represented the pellet fraction (insoluble) and supernatant fraction (soluble), respectively. The oval shapes highlight the bands of dxs and tal proteins. Protein tal and dxs were expressed in K3 medium with 20 g/L glucose at 30 °C. ( b) Quantitative presentation of the SDS-PAGE images in a . The protein solubility was the ratio of soluble protein amount to the total protein amount. The protein amount was estimated by using band intensity on SDS-PAGE images. The sequences of the designed tags for N-terminal and C-terminal were shown. The amino acid S and G on the two ends of the tags were the linkers for GT DNA assembly standard, which was used to construct the plasmids in this study 40 .

    Techniques Used: SDS Page, Solubility, Construct

    3) Product Images from "Improve Protein Solubility and Activity based on Machine Learning Models"

    Article Title: Improve Protein Solubility and Activity based on Machine Learning Models

    Journal: bioRxiv

    doi: 10.1101/817890

    ( a) The SDS-PAGE analysis of protein tal and dxs expressed in E. coli with and without tags designed by our optimization algorithm. “+” and “-” represented expressed proteins with and without peptide tags respectively. “P” and “S” represented the pellet fraction (insoluble) and supernatant fraction (soluble), respectively. The oval shapes highlight the bands of dxs and tal proteins. Protein tal and dxs were expressed in K3 medium with 20 g/L glucose at 30 °C. ( b) Quantitative presentation of the SDS-PAGE images in a . The protein solubility was the ratio of soluble protein amount to the total protein amount. The protein amount was estimated by using band intensity on SDS-PAGE images. The sequences of the designed tags for N-terminal and C-terminal were shown. The amino acid S and G on the two ends of the tags were the linkers for GT DNA assembly standard, which was used to construct the plasmids in this study 40 .
    Figure Legend Snippet: ( a) The SDS-PAGE analysis of protein tal and dxs expressed in E. coli with and without tags designed by our optimization algorithm. “+” and “-” represented expressed proteins with and without peptide tags respectively. “P” and “S” represented the pellet fraction (insoluble) and supernatant fraction (soluble), respectively. The oval shapes highlight the bands of dxs and tal proteins. Protein tal and dxs were expressed in K3 medium with 20 g/L glucose at 30 °C. ( b) Quantitative presentation of the SDS-PAGE images in a . The protein solubility was the ratio of soluble protein amount to the total protein amount. The protein amount was estimated by using band intensity on SDS-PAGE images. The sequences of the designed tags for N-terminal and C-terminal were shown. The amino acid S and G on the two ends of the tags were the linkers for GT DNA assembly standard, which was used to construct the plasmids in this study 40 .

    Techniques Used: SDS Page, Solubility, Construct

    4) Product Images from "Towards improving proximity labeling by the biotin ligase BirA"

    Article Title: Towards improving proximity labeling by the biotin ligase BirA

    Journal: Methods (San Diego, Calif.)

    doi: 10.1016/j.ymeth.2018.11.003

    Evaluation of auto-biotinylation by mutants of BirA at position 118. (A) Purification and biotinylation state (fl-neutra detection) of wild-type and mutant BirA proteins expressed as MBP fusions in E. coli . Each mutant protein contains a single amino acid substitution at position 118, as indicated. Equal amounts (1 μg) of each MBP-BirA mutant were analyzed by Coomassie blue staining and by blotting. (B) Scheme for Factor Xa mediated cleavage and analysis of auto-biotinylation. (C) Biotin detection within the MBP and BirA catalytic fragments of each BirA mutant (2.5 μg) before and after cleavage with Factor Xa. Blots were also probed with anti-MBP antibody. Bands corresponding to MBP-BirA, MBP, and BirA are indicated to the right of each panel. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
    Figure Legend Snippet: Evaluation of auto-biotinylation by mutants of BirA at position 118. (A) Purification and biotinylation state (fl-neutra detection) of wild-type and mutant BirA proteins expressed as MBP fusions in E. coli . Each mutant protein contains a single amino acid substitution at position 118, as indicated. Equal amounts (1 μg) of each MBP-BirA mutant were analyzed by Coomassie blue staining and by blotting. (B) Scheme for Factor Xa mediated cleavage and analysis of auto-biotinylation. (C) Biotin detection within the MBP and BirA catalytic fragments of each BirA mutant (2.5 μg) before and after cleavage with Factor Xa. Blots were also probed with anti-MBP antibody. Bands corresponding to MBP-BirA, MBP, and BirA are indicated to the right of each panel. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

    Techniques Used: Purification, Mutagenesis, Staining

    Bead-based analysis of proximal biotinylation by BirA mutants. (A) Scheme for proximal (self and trans) biotinylation by BirA immobilized on an amylose bead surface. (B) Biotinylation reactions were performed with MBP-BirA mutants (5 μg) and free MPB (10 μg) on amylose beads in the presence of biotin (50 μM). Samples were analyzed by fl-neutra. Normalized biotinylation of self and trans labeling was quantified and plotted in (C).
    Figure Legend Snippet: Bead-based analysis of proximal biotinylation by BirA mutants. (A) Scheme for proximal (self and trans) biotinylation by BirA immobilized on an amylose bead surface. (B) Biotinylation reactions were performed with MBP-BirA mutants (5 μg) and free MPB (10 μg) on amylose beads in the presence of biotin (50 μM). Samples were analyzed by fl-neutra. Normalized biotinylation of self and trans labeling was quantified and plotted in (C).

    Techniques Used: Labeling

    Utilization of Biotin Acceptor Peptide (BAPs) for analyzing BirA mutants. (A) BAP sequences showing the position of the acceptor lysine and amino acid substitutions (bold underline). (B) In vitro biotinylation of BAP substrates with purified BirA enzymes. Reactions contained MBP-BirA (1 μg) with the indicated amount of each BAP and biotin (50 μM). (C) Quantification of relative biotinylation of GST-BAP (K- > A) in (B) indicates proximity labeling of a site outside of the BAP acceptor lysine. (D) TLC analysis of basal and BAP-induced bioAMP and AMP generation by BirA proteins. Assays were performed in triplicate and analyzed by autoradiography, one set of which is shown in this panel. (E) Quantification of AMP generation by BirA mutants. The % AMP is the percentage of signal in each lane corresponding to [α- 32 P]-labeled AMP. (****p
    Figure Legend Snippet: Utilization of Biotin Acceptor Peptide (BAPs) for analyzing BirA mutants. (A) BAP sequences showing the position of the acceptor lysine and amino acid substitutions (bold underline). (B) In vitro biotinylation of BAP substrates with purified BirA enzymes. Reactions contained MBP-BirA (1 μg) with the indicated amount of each BAP and biotin (50 μM). (C) Quantification of relative biotinylation of GST-BAP (K- > A) in (B) indicates proximity labeling of a site outside of the BAP acceptor lysine. (D) TLC analysis of basal and BAP-induced bioAMP and AMP generation by BirA proteins. Assays were performed in triplicate and analyzed by autoradiography, one set of which is shown in this panel. (E) Quantification of AMP generation by BirA mutants. The % AMP is the percentage of signal in each lane corresponding to [α- 32 P]-labeled AMP. (****p

    Techniques Used: In Vitro, Purification, Labeling, Thin Layer Chromatography, Autoradiography

    5) Product Images from "Pan-Serotype Diagnostic for Foot-and-Mouth Disease Using the Consensus Antigen of Nonstructural Protein 3B"

    Article Title: Pan-Serotype Diagnostic for Foot-and-Mouth Disease Using the Consensus Antigen of Nonstructural Protein 3B

    Journal: Journal of Clinical Microbiology

    doi: 10.1128/JCM.03491-14

    Recombinant c3B protein expression, purification, and detection. (A) Coomassie blue-stained polyacrylamide gel of recombinant MBP-c3B protein expressed in BL21  E. coli  and affinity purified using amylose resin. Lane M, molecular mass markers (Mark 12;
    Figure Legend Snippet: Recombinant c3B protein expression, purification, and detection. (A) Coomassie blue-stained polyacrylamide gel of recombinant MBP-c3B protein expressed in BL21 E. coli and affinity purified using amylose resin. Lane M, molecular mass markers (Mark 12;

    Techniques Used: Recombinant, Expressing, Purification, Staining, Affinity Purification

    Related Articles

    Centrifugation:

    Article Title: A Cyan Fluorescent Reporter Expressed from the Chloroplast Genome of Marchantia polymorpha
    Article Snippet: .. Visualization of proteins by in-gel fluorescence and coomassie staining For total protein extraction from M. polymorpha tissues, 50 mg (fresh weight) of thallus were ground by liquid nitrogen and the resulting powder vortexed in 200 µl of 2 × Tris–glycine for 30 s. For total protein extraction from BL21 or T7 Express competent E. coli (New England Biolabs), an aliquot of overnight culture containing approximately 0.5–1 × 109 cells was pelleted by centrifugation at 14,000 r.p.m. and 4°C for 1 min, resuspended in 50 µl of 2 × Tris–glycine SDS sample buffer and vortexed for 30 s. In both cases, 2-fold dilutions of crude extracts were centrifuged at 14,000 r.p.m. and 4°C for 20 min to remove cell debris, and unboiled (unless indicated otherwise) supernatants were directly loaded onto a NuPAGE Novex 4–12% Bis–Tris protein minigel (Life Technologies). .. Sample proteins were separated by SDS–PAGE in MES SDS running buffer over 35 min at 200 V, and the gel subsequently was shaken in dH2 O for 3 × 5 min to remove excess SDS and buffer salts.

    Over Expression:

    Article Title: Pan-Serotype Diagnostic for Foot-and-Mouth Disease Using the Consensus Antigen of Nonstructural Protein 3B
    Article Snippet: .. Chemically competent BL21 E. coli (NEB) was transformed with 10 ng pMAL-c5e or pMAL-c3B for overexpression of recombinant MBP or MBP-c3B fusion protein, according to the manufacturer's instructions. .. Cultures of each clone were expanded to 100 ml in Luria-Bertani broth containing 100 μg/ml ampicillin and 0.2% (wt/vol) glucose by incubation at 37°C, with shaking at 225 rpm in an orbital shaking incubator, until the optical density at 600 nm (OD600 ) reached 0.5 to 0.6 units.

    Fluorescence:

    Article Title: A Cyan Fluorescent Reporter Expressed from the Chloroplast Genome of Marchantia polymorpha
    Article Snippet: .. Visualization of proteins by in-gel fluorescence and coomassie staining For total protein extraction from M. polymorpha tissues, 50 mg (fresh weight) of thallus were ground by liquid nitrogen and the resulting powder vortexed in 200 µl of 2 × Tris–glycine for 30 s. For total protein extraction from BL21 or T7 Express competent E. coli (New England Biolabs), an aliquot of overnight culture containing approximately 0.5–1 × 109 cells was pelleted by centrifugation at 14,000 r.p.m. and 4°C for 1 min, resuspended in 50 µl of 2 × Tris–glycine SDS sample buffer and vortexed for 30 s. In both cases, 2-fold dilutions of crude extracts were centrifuged at 14,000 r.p.m. and 4°C for 20 min to remove cell debris, and unboiled (unless indicated otherwise) supernatants were directly loaded onto a NuPAGE Novex 4–12% Bis–Tris protein minigel (Life Technologies). .. Sample proteins were separated by SDS–PAGE in MES SDS running buffer over 35 min at 200 V, and the gel subsequently was shaken in dH2 O for 3 × 5 min to remove excess SDS and buffer salts.

    Cell Culture:

    Article Title: Ubiquitin-specific peptidase 2a (USP2a) deubiquitinates and stabilizes β-catenin
    Article Snippet: .. BL21 competent cells (NEB) were transformed with GST-GFP, GST-USP2a, or GST-USP2aC276A and cultured in LB medium. .. GST fusion proteins were induced by IPTG (Ambion).

    Article Title: Improve Protein Solubility and Activity based on Machine Learning Models
    Article Snippet: .. Cell culture and SDS-PAGE analysis of protein solubility Each of constructed plasmid was introduced into E. coli BL21 (DE3) (C2530H, New England Biolabs) for SDS-PAGE analysis by using standard heat shock protocol. .. In order to test the resulting strains, single colony was inoculated into 1 mL of LB with 100 µg/mL of ampicillin, and was cultured overnight at 37 °C/250 rpm.

    Purification:

    Article Title: Cell Death Suppressor Arabidopsis Bax Inhibitor-1 Is Associated with Calmodulin Binding and Ion Homeostasis 1Cell Death Suppressor Arabidopsis Bax Inhibitor-1 Is Associated with Calmodulin Binding and Ion Homeostasis 1 [OA]
    Article Snippet: .. The resultant vector was transformed into E. coli BL21 strain and MBP-tagged BI-C protein was purified according to the instructions provided by the manufacturer (NEB). .. The empty pMAL vector possessing in-frame Gal was used as a control (MAL-Gal).

    Protein Extraction:

    Article Title: A Cyan Fluorescent Reporter Expressed from the Chloroplast Genome of Marchantia polymorpha
    Article Snippet: .. Visualization of proteins by in-gel fluorescence and coomassie staining For total protein extraction from M. polymorpha tissues, 50 mg (fresh weight) of thallus were ground by liquid nitrogen and the resulting powder vortexed in 200 µl of 2 × Tris–glycine for 30 s. For total protein extraction from BL21 or T7 Express competent E. coli (New England Biolabs), an aliquot of overnight culture containing approximately 0.5–1 × 109 cells was pelleted by centrifugation at 14,000 r.p.m. and 4°C for 1 min, resuspended in 50 µl of 2 × Tris–glycine SDS sample buffer and vortexed for 30 s. In both cases, 2-fold dilutions of crude extracts were centrifuged at 14,000 r.p.m. and 4°C for 20 min to remove cell debris, and unboiled (unless indicated otherwise) supernatants were directly loaded onto a NuPAGE Novex 4–12% Bis–Tris protein minigel (Life Technologies). .. Sample proteins were separated by SDS–PAGE in MES SDS running buffer over 35 min at 200 V, and the gel subsequently was shaken in dH2 O for 3 × 5 min to remove excess SDS and buffer salts.

    Construct:

    Article Title: Improve Protein Solubility and Activity based on Machine Learning Models
    Article Snippet: .. Cell culture and SDS-PAGE analysis of protein solubility Each of constructed plasmid was introduced into E. coli BL21 (DE3) (C2530H, New England Biolabs) for SDS-PAGE analysis by using standard heat shock protocol. .. In order to test the resulting strains, single colony was inoculated into 1 mL of LB with 100 µg/mL of ampicillin, and was cultured overnight at 37 °C/250 rpm.

    Staining:

    Article Title: A Cyan Fluorescent Reporter Expressed from the Chloroplast Genome of Marchantia polymorpha
    Article Snippet: .. Visualization of proteins by in-gel fluorescence and coomassie staining For total protein extraction from M. polymorpha tissues, 50 mg (fresh weight) of thallus were ground by liquid nitrogen and the resulting powder vortexed in 200 µl of 2 × Tris–glycine for 30 s. For total protein extraction from BL21 or T7 Express competent E. coli (New England Biolabs), an aliquot of overnight culture containing approximately 0.5–1 × 109 cells was pelleted by centrifugation at 14,000 r.p.m. and 4°C for 1 min, resuspended in 50 µl of 2 × Tris–glycine SDS sample buffer and vortexed for 30 s. In both cases, 2-fold dilutions of crude extracts were centrifuged at 14,000 r.p.m. and 4°C for 20 min to remove cell debris, and unboiled (unless indicated otherwise) supernatants were directly loaded onto a NuPAGE Novex 4–12% Bis–Tris protein minigel (Life Technologies). .. Sample proteins were separated by SDS–PAGE in MES SDS running buffer over 35 min at 200 V, and the gel subsequently was shaken in dH2 O for 3 × 5 min to remove excess SDS and buffer salts.

    Transformation Assay:

    Article Title: Evaluation and minimization of Cas9-independent off-target DNA editing by cytosine base editors.
    Article Snippet: .. Cytosine base editors (CBEs) enable targeted C•G-to-T•A conversions in genomic DNA. .. Cytosine base editors (CBEs) enable targeted C•G-to-T•A conversions in genomic DNA.

    Article Title: Ubiquitin-specific peptidase 2a (USP2a) deubiquitinates and stabilizes β-catenin
    Article Snippet: .. BL21 competent cells (NEB) were transformed with GST-GFP, GST-USP2a, or GST-USP2aC276A and cultured in LB medium. .. GST fusion proteins were induced by IPTG (Ambion).

    Article Title: Knockout of a difficult-to-remove CHO host cell protein, lipoprotein lipase, for improved polysorbate stability in monoclonal antibody formulations
    Article Snippet: .. The lpl -containing pET11a plasmid was then transformed into BL21 competent cells in SOC broth (New England BioLabs) and plated on ampicillin. ..

    Article Title: Pan-Serotype Diagnostic for Foot-and-Mouth Disease Using the Consensus Antigen of Nonstructural Protein 3B
    Article Snippet: .. Chemically competent BL21 E. coli (NEB) was transformed with 10 ng pMAL-c5e or pMAL-c3B for overexpression of recombinant MBP or MBP-c3B fusion protein, according to the manufacturer's instructions. .. Cultures of each clone were expanded to 100 ml in Luria-Bertani broth containing 100 μg/ml ampicillin and 0.2% (wt/vol) glucose by incubation at 37°C, with shaking at 225 rpm in an orbital shaking incubator, until the optical density at 600 nm (OD600 ) reached 0.5 to 0.6 units.

    Article Title: Cell Death Suppressor Arabidopsis Bax Inhibitor-1 Is Associated with Calmodulin Binding and Ion Homeostasis 1Cell Death Suppressor Arabidopsis Bax Inhibitor-1 Is Associated with Calmodulin Binding and Ion Homeostasis 1 [OA]
    Article Snippet: .. The resultant vector was transformed into E. coli BL21 strain and MBP-tagged BI-C protein was purified according to the instructions provided by the manufacturer (NEB). .. The empty pMAL vector possessing in-frame Gal was used as a control (MAL-Gal).

    Recombinant:

    Article Title: Pan-Serotype Diagnostic for Foot-and-Mouth Disease Using the Consensus Antigen of Nonstructural Protein 3B
    Article Snippet: .. Chemically competent BL21 E. coli (NEB) was transformed with 10 ng pMAL-c5e or pMAL-c3B for overexpression of recombinant MBP or MBP-c3B fusion protein, according to the manufacturer's instructions. .. Cultures of each clone were expanded to 100 ml in Luria-Bertani broth containing 100 μg/ml ampicillin and 0.2% (wt/vol) glucose by incubation at 37°C, with shaking at 225 rpm in an orbital shaking incubator, until the optical density at 600 nm (OD600 ) reached 0.5 to 0.6 units.

    SDS Page:

    Article Title: Improve Protein Solubility and Activity based on Machine Learning Models
    Article Snippet: .. Cell culture and SDS-PAGE analysis of protein solubility Each of constructed plasmid was introduced into E. coli BL21 (DE3) (C2530H, New England Biolabs) for SDS-PAGE analysis by using standard heat shock protocol. .. In order to test the resulting strains, single colony was inoculated into 1 mL of LB with 100 µg/mL of ampicillin, and was cultured overnight at 37 °C/250 rpm.

    Plasmid Preparation:

    Article Title: Evaluation and minimization of Cas9-independent off-target DNA editing by cytosine base editors.
    Article Snippet: .. Cytosine base editors (CBEs) enable targeted C•G-to-T•A conversions in genomic DNA. .. Cytosine base editors (CBEs) enable targeted C•G-to-T•A conversions in genomic DNA.

    Article Title: Knockout of a difficult-to-remove CHO host cell protein, lipoprotein lipase, for improved polysorbate stability in monoclonal antibody formulations
    Article Snippet: .. The lpl -containing pET11a plasmid was then transformed into BL21 competent cells in SOC broth (New England BioLabs) and plated on ampicillin. ..

    Article Title: Improve Protein Solubility and Activity based on Machine Learning Models
    Article Snippet: .. Cell culture and SDS-PAGE analysis of protein solubility Each of constructed plasmid was introduced into E. coli BL21 (DE3) (C2530H, New England Biolabs) for SDS-PAGE analysis by using standard heat shock protocol. .. In order to test the resulting strains, single colony was inoculated into 1 mL of LB with 100 µg/mL of ampicillin, and was cultured overnight at 37 °C/250 rpm.

    Article Title: Cell Death Suppressor Arabidopsis Bax Inhibitor-1 Is Associated with Calmodulin Binding and Ion Homeostasis 1Cell Death Suppressor Arabidopsis Bax Inhibitor-1 Is Associated with Calmodulin Binding and Ion Homeostasis 1 [OA]
    Article Snippet: .. The resultant vector was transformed into E. coli BL21 strain and MBP-tagged BI-C protein was purified according to the instructions provided by the manufacturer (NEB). .. The empty pMAL vector possessing in-frame Gal was used as a control (MAL-Gal).

    Solubility:

    Article Title: Improve Protein Solubility and Activity based on Machine Learning Models
    Article Snippet: .. Cell culture and SDS-PAGE analysis of protein solubility Each of constructed plasmid was introduced into E. coli BL21 (DE3) (C2530H, New England Biolabs) for SDS-PAGE analysis by using standard heat shock protocol. .. In order to test the resulting strains, single colony was inoculated into 1 mL of LB with 100 µg/mL of ampicillin, and was cultured overnight at 37 °C/250 rpm.

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    New England Biolabs bl21
    Detection of chloroplast-expressed mTurquoise2 in transgenic lines of Marchantia polymorpha. (A) M. polymorpha protein extracts separated by SDS–PAGE. Protein extracts from wild type (WT) and transplastomic (CL0*b) lines of M. polymorpha were separated by 4–12% SDS–PAGE, and visualized by in-gel fluorescence (left) and subsequent Coomassie stain (right). The fluorescence image was generated using a custom imaging device described in the Materials and Methods for visualization of CFP bands (emission 486/10 nm, green) and marker (emission 540/10 nm, red). (B) Untagged and His 6 -tagged mTurquoise2 separated by SDS–PAGE. Untagged mTurquoise2 was expressed from plasmids pCS CL0*b ( psbA promoter) in <t>BL21</t> E. coli and pCRB SREI (T7 promoter) in T7 Express E. coli. His 6 -tagged mTurquoise2 was expressed from plasmid pCRB SREI 6his (T7 promoter) and purified by affinity chromatography. The rightmost lanes contain 50 ng of unboiled and boiled purified protein, respectively. Visualization by in-gel fluorescence and Coomassie stain was conducted as described above. (C) Standard curve for quantification of mTurquoise2 based on in-gel fluorescence. Serial dilutions of 1 µg purified of mTurquoise2 were separated by 4–12% SDS–PAGE and the band intensity quantified by in-gel fluorescence as described in the Materials and Methods. Levels of mTurquoise2 extracted from transplastomic (CL0*b) M. polymorpha (red cross) were estimated by linear regression analysis on densities of fluorescent target bands as shown in (A). Error bars represent the SD of average band density between three different experiments.
    Bl21, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 126 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Detection of chloroplast-expressed mTurquoise2 in transgenic lines of Marchantia polymorpha. (A) M. polymorpha protein extracts separated by SDS–PAGE. Protein extracts from wild type (WT) and transplastomic (CL0*b) lines of M. polymorpha were separated by 4–12% SDS–PAGE, and visualized by in-gel fluorescence (left) and subsequent Coomassie stain (right). The fluorescence image was generated using a custom imaging device described in the Materials and Methods for visualization of CFP bands (emission 486/10 nm, green) and marker (emission 540/10 nm, red). (B) Untagged and His 6 -tagged mTurquoise2 separated by SDS–PAGE. Untagged mTurquoise2 was expressed from plasmids pCS CL0*b ( psbA promoter) in BL21 E. coli and pCRB SREI (T7 promoter) in T7 Express E. coli. His 6 -tagged mTurquoise2 was expressed from plasmid pCRB SREI 6his (T7 promoter) and purified by affinity chromatography. The rightmost lanes contain 50 ng of unboiled and boiled purified protein, respectively. Visualization by in-gel fluorescence and Coomassie stain was conducted as described above. (C) Standard curve for quantification of mTurquoise2 based on in-gel fluorescence. Serial dilutions of 1 µg purified of mTurquoise2 were separated by 4–12% SDS–PAGE and the band intensity quantified by in-gel fluorescence as described in the Materials and Methods. Levels of mTurquoise2 extracted from transplastomic (CL0*b) M. polymorpha (red cross) were estimated by linear regression analysis on densities of fluorescent target bands as shown in (A). Error bars represent the SD of average band density between three different experiments.

    Journal: Plant and Cell Physiology

    Article Title: A Cyan Fluorescent Reporter Expressed from the Chloroplast Genome of Marchantia polymorpha

    doi: 10.1093/pcp/pcv160

    Figure Lengend Snippet: Detection of chloroplast-expressed mTurquoise2 in transgenic lines of Marchantia polymorpha. (A) M. polymorpha protein extracts separated by SDS–PAGE. Protein extracts from wild type (WT) and transplastomic (CL0*b) lines of M. polymorpha were separated by 4–12% SDS–PAGE, and visualized by in-gel fluorescence (left) and subsequent Coomassie stain (right). The fluorescence image was generated using a custom imaging device described in the Materials and Methods for visualization of CFP bands (emission 486/10 nm, green) and marker (emission 540/10 nm, red). (B) Untagged and His 6 -tagged mTurquoise2 separated by SDS–PAGE. Untagged mTurquoise2 was expressed from plasmids pCS CL0*b ( psbA promoter) in BL21 E. coli and pCRB SREI (T7 promoter) in T7 Express E. coli. His 6 -tagged mTurquoise2 was expressed from plasmid pCRB SREI 6his (T7 promoter) and purified by affinity chromatography. The rightmost lanes contain 50 ng of unboiled and boiled purified protein, respectively. Visualization by in-gel fluorescence and Coomassie stain was conducted as described above. (C) Standard curve for quantification of mTurquoise2 based on in-gel fluorescence. Serial dilutions of 1 µg purified of mTurquoise2 were separated by 4–12% SDS–PAGE and the band intensity quantified by in-gel fluorescence as described in the Materials and Methods. Levels of mTurquoise2 extracted from transplastomic (CL0*b) M. polymorpha (red cross) were estimated by linear regression analysis on densities of fluorescent target bands as shown in (A). Error bars represent the SD of average band density between three different experiments.

    Article Snippet: Visualization of proteins by in-gel fluorescence and coomassie staining For total protein extraction from M. polymorpha tissues, 50 mg (fresh weight) of thallus were ground by liquid nitrogen and the resulting powder vortexed in 200 µl of 2 × Tris–glycine for 30 s. For total protein extraction from BL21 or T7 Express competent E. coli (New England Biolabs), an aliquot of overnight culture containing approximately 0.5–1 × 109 cells was pelleted by centrifugation at 14,000 r.p.m. and 4°C for 1 min, resuspended in 50 µl of 2 × Tris–glycine SDS sample buffer and vortexed for 30 s. In both cases, 2-fold dilutions of crude extracts were centrifuged at 14,000 r.p.m. and 4°C for 20 min to remove cell debris, and unboiled (unless indicated otherwise) supernatants were directly loaded onto a NuPAGE Novex 4–12% Bis–Tris protein minigel (Life Technologies).

    Techniques: Transgenic Assay, SDS Page, Fluorescence, Staining, Generated, Imaging, Marker, Plasmid Preparation, Purification, Affinity Chromatography

    ( a) The SDS-PAGE analysis of protein tal and dxs expressed in E. coli with and without tags designed by our optimization algorithm. “+” and “-” represented expressed proteins with and without peptide tags respectively. “P” and “S” represented the pellet fraction (insoluble) and supernatant fraction (soluble), respectively. The oval shapes highlight the bands of dxs and tal proteins. Protein tal and dxs were expressed in K3 medium with 20 g/L glucose at 30 °C. ( b) Quantitative presentation of the SDS-PAGE images in a . The protein solubility was the ratio of soluble protein amount to the total protein amount. The protein amount was estimated by using band intensity on SDS-PAGE images. The sequences of the designed tags for N-terminal and C-terminal were shown. The amino acid S and G on the two ends of the tags were the linkers for GT DNA assembly standard, which was used to construct the plasmids in this study 40 .

    Journal: bioRxiv

    Article Title: Improve Protein Solubility and Activity based on Machine Learning Models

    doi: 10.1101/817890

    Figure Lengend Snippet: ( a) The SDS-PAGE analysis of protein tal and dxs expressed in E. coli with and without tags designed by our optimization algorithm. “+” and “-” represented expressed proteins with and without peptide tags respectively. “P” and “S” represented the pellet fraction (insoluble) and supernatant fraction (soluble), respectively. The oval shapes highlight the bands of dxs and tal proteins. Protein tal and dxs were expressed in K3 medium with 20 g/L glucose at 30 °C. ( b) Quantitative presentation of the SDS-PAGE images in a . The protein solubility was the ratio of soluble protein amount to the total protein amount. The protein amount was estimated by using band intensity on SDS-PAGE images. The sequences of the designed tags for N-terminal and C-terminal were shown. The amino acid S and G on the two ends of the tags were the linkers for GT DNA assembly standard, which was used to construct the plasmids in this study 40 .

    Article Snippet: Cell culture and SDS-PAGE analysis of protein solubility Each of constructed plasmid was introduced into E. coli BL21 (DE3) (C2530H, New England Biolabs) for SDS-PAGE analysis by using standard heat shock protocol.

    Techniques: SDS Page, Solubility, Construct

    Recombinant c3B protein expression, purification, and detection. (A) Coomassie blue-stained polyacrylamide gel of recombinant MBP-c3B protein expressed in BL21  E. coli  and affinity purified using amylose resin. Lane M, molecular mass markers (Mark 12;

    Journal: Journal of Clinical Microbiology

    Article Title: Pan-Serotype Diagnostic for Foot-and-Mouth Disease Using the Consensus Antigen of Nonstructural Protein 3B

    doi: 10.1128/JCM.03491-14

    Figure Lengend Snippet: Recombinant c3B protein expression, purification, and detection. (A) Coomassie blue-stained polyacrylamide gel of recombinant MBP-c3B protein expressed in BL21 E. coli and affinity purified using amylose resin. Lane M, molecular mass markers (Mark 12;

    Article Snippet: Chemically competent BL21 E. coli (NEB) was transformed with 10 ng pMAL-c5e or pMAL-c3B for overexpression of recombinant MBP or MBP-c3B fusion protein, according to the manufacturer's instructions.

    Techniques: Recombinant, Expressing, Purification, Staining, Affinity Purification