Structured Review

Thermo Fisher bl21
The expression and purification of recombinant CAV viral proteins. The three expression plasmids showed in Figure 1 were express in E. coli <t>BL21</t> (DE3) and purified by GST affinity chromatography. The recombinant GST-tag proteins were separated by SDS-PAGE (a) and detected by Western-blotting (b) . Anti-GST tag monoclonal antibody was used to recognize the recombinant viral proteins. Lane M, pre-stained protein marker; lane BI, before IPTG induction; lane AI, after IPTG induction and cultivation for 4 hr; lane P, purified recombinant GST-tag protein. The red solid triangles pinpoint the various proteins.
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Images

1) Product Images from "Expression and characterization of highly antigenic domains of chicken anemia virus viral VP2 and VP3 subunit proteins in a recombinant E. coli for sero-diagnostic applications"

Article Title: Expression and characterization of highly antigenic domains of chicken anemia virus viral VP2 and VP3 subunit proteins in a recombinant E. coli for sero-diagnostic applications

Journal: BMC Veterinary Research

doi: 10.1186/1746-6148-9-161

The expression and purification of recombinant CAV viral proteins. The three expression plasmids showed in Figure 1 were express in E. coli BL21 (DE3) and purified by GST affinity chromatography. The recombinant GST-tag proteins were separated by SDS-PAGE (a) and detected by Western-blotting (b) . Anti-GST tag monoclonal antibody was used to recognize the recombinant viral proteins. Lane M, pre-stained protein marker; lane BI, before IPTG induction; lane AI, after IPTG induction and cultivation for 4 hr; lane P, purified recombinant GST-tag protein. The red solid triangles pinpoint the various proteins.
Figure Legend Snippet: The expression and purification of recombinant CAV viral proteins. The three expression plasmids showed in Figure 1 were express in E. coli BL21 (DE3) and purified by GST affinity chromatography. The recombinant GST-tag proteins were separated by SDS-PAGE (a) and detected by Western-blotting (b) . Anti-GST tag monoclonal antibody was used to recognize the recombinant viral proteins. Lane M, pre-stained protein marker; lane BI, before IPTG induction; lane AI, after IPTG induction and cultivation for 4 hr; lane P, purified recombinant GST-tag protein. The red solid triangles pinpoint the various proteins.

Techniques Used: Expressing, Purification, Recombinant, Affinity Chromatography, SDS Page, Western Blot, Staining, Marker

The expression and purification results for the five recombinant VP2 and three recombinant VP3 subunit proteins. All subunit proteins were purified by GST affinity chromatography after expression in E. coli BL21 (DE3). The presence of the protein was detected by SDS-PAGE (a) and by Western-blotting (b) . Anti-GST tag monoclonal antibody was used to recognize the VP2 and VP3 subunit proteins when the various different expression plasmids, as described in Figure 1 , were used. Lane M, pre-stained protein marker; lane 1, 4, 7, 10, 13, 16, 19 and 22, before IPTG induction; lane 2, 5, 8, 11, 14, 17, 20 and 23, after IPTG induction and 4 h cultivation; lane 3, 6, 9, 12, 15, 18, 21 and, 24 after purification by GST affinity chromatography. The solid triangles pinpoint the expressed and purified VP2 and VP3 subunit proteins, respectively.
Figure Legend Snippet: The expression and purification results for the five recombinant VP2 and three recombinant VP3 subunit proteins. All subunit proteins were purified by GST affinity chromatography after expression in E. coli BL21 (DE3). The presence of the protein was detected by SDS-PAGE (a) and by Western-blotting (b) . Anti-GST tag monoclonal antibody was used to recognize the VP2 and VP3 subunit proteins when the various different expression plasmids, as described in Figure 1 , were used. Lane M, pre-stained protein marker; lane 1, 4, 7, 10, 13, 16, 19 and 22, before IPTG induction; lane 2, 5, 8, 11, 14, 17, 20 and 23, after IPTG induction and 4 h cultivation; lane 3, 6, 9, 12, 15, 18, 21 and, 24 after purification by GST affinity chromatography. The solid triangles pinpoint the expressed and purified VP2 and VP3 subunit proteins, respectively.

Techniques Used: Expressing, Purification, Recombinant, Affinity Chromatography, SDS Page, Western Blot, Staining, Marker

2) Product Images from "Development of New Recombinant DgK Antigen for Diagnosis of Dirofilaria immitis Infections in Dogs Using ELISA Technique and Its Comparison to Molecular Methods"

Article Title: Development of New Recombinant DgK Antigen for Diagnosis of Dirofilaria immitis Infections in Dogs Using ELISA Technique and Its Comparison to Molecular Methods

Journal: Iranian Biomedical Journal

doi: 10.22034/ibj.22.4.283

SDS-PAGE analysis of purified antigen and Western blot analysis of rDgK antigen. (a) Purified protein was loaded on 12% acrylamide gel, stained with Coomassie brilliant blue R-250. Lane M, protein molecular standard; lane 1, SDS-PAGE of purified recombinant protein (arrow). (b) Lane M, protein molecular standard; lane 1, Western blotting of expressed rDgK probed with (lane 1) the negative control pool sera, (lane 2) suspected dog pool sera, and (lane 3) the positive-control serum pools (arrows). Lane 4 indicates total protein of untransformed E. coli BL21 in Western blot with the positive-control serum pools.
Figure Legend Snippet: SDS-PAGE analysis of purified antigen and Western blot analysis of rDgK antigen. (a) Purified protein was loaded on 12% acrylamide gel, stained with Coomassie brilliant blue R-250. Lane M, protein molecular standard; lane 1, SDS-PAGE of purified recombinant protein (arrow). (b) Lane M, protein molecular standard; lane 1, Western blotting of expressed rDgK probed with (lane 1) the negative control pool sera, (lane 2) suspected dog pool sera, and (lane 3) the positive-control serum pools (arrows). Lane 4 indicates total protein of untransformed E. coli BL21 in Western blot with the positive-control serum pools.

Techniques Used: SDS Page, Purification, Western Blot, Acrylamide Gel Assay, Staining, Recombinant, Negative Control, Positive Control

3) Product Images from "Production of Active Nonglycosylated Recombinant B-Chain of Type-2 Ribosome-Inactivating Protein from Viscum articulatum and Its Biological Effects on Peripheral Blood Mononuclear Cells"

Article Title: Production of Active Nonglycosylated Recombinant B-Chain of Type-2 Ribosome-Inactivating Protein from Viscum articulatum and Its Biological Effects on Peripheral Blood Mononuclear Cells

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

doi: 10.1155/2011/283747

SDS-PAGE (12.5% polyacrylamide gel) of rATB. Lanes: 1: molecular size markers; 2: E. coli BL21 (pET28arATB) prior to induction; 3: E. coli BL21 (pET28arATB) after induction; 4: 5 μ g denatured rATB in inclusion bodies; 5: 1 μ g refolded soluble rATB.
Figure Legend Snippet: SDS-PAGE (12.5% polyacrylamide gel) of rATB. Lanes: 1: molecular size markers; 2: E. coli BL21 (pET28arATB) prior to induction; 3: E. coli BL21 (pET28arATB) after induction; 4: 5 μ g denatured rATB in inclusion bodies; 5: 1 μ g refolded soluble rATB.

Techniques Used: SDS Page

4) Product Images from "A Bacterial Surface Display System Expressing Cleavable Capsid Proteins of Human Norovirus: A Novel System to Discover Candidate Receptors"

Article Title: A Bacterial Surface Display System Expressing Cleavable Capsid Proteins of Human Norovirus: A Novel System to Discover Candidate Receptors

Journal: Frontiers in Microbiology

doi: 10.3389/fmicb.2017.02405

SDS-PAGE (A) and Western Blot (B) analysis of the expression of E. coli cell surface display systems for HuNoV (GII.4) recombinant capsid proteins with (3) or without the linker fragment (2) and the thrombin-released P proteins (4). M: prestained protein ladder (Catalog No.: 26616, Thermo Fisher, Shanghai, China); (1) pET-28a/BL21; (2) pET28a-inaQn-P (GII.4)/BL21; (3) pET28a-inaQn-TB-P (GII.4) /BL21; (4) Thrombin-released P proteins.
Figure Legend Snippet: SDS-PAGE (A) and Western Blot (B) analysis of the expression of E. coli cell surface display systems for HuNoV (GII.4) recombinant capsid proteins with (3) or without the linker fragment (2) and the thrombin-released P proteins (4). M: prestained protein ladder (Catalog No.: 26616, Thermo Fisher, Shanghai, China); (1) pET-28a/BL21; (2) pET28a-inaQn-P (GII.4)/BL21; (3) pET28a-inaQn-TB-P (GII.4) /BL21; (4) Thrombin-released P proteins.

Techniques Used: SDS Page, Western Blot, Expressing, Recombinant, Positron Emission Tomography

OD of type A HBGA bound to serially diluted thrombin-released P proteins (red) and the thrombin-released background proteins from pET28a-inaQn-TB/BL21 (black). Bars represent standard errors.
Figure Legend Snippet: OD of type A HBGA bound to serially diluted thrombin-released P proteins (red) and the thrombin-released background proteins from pET28a-inaQn-TB/BL21 (black). Bars represent standard errors.

Techniques Used:

5) Product Images from "Immunogenicity and protective efficacy of recombinant alkaline shock protein 23 from Staphylococcus aureus in a murine model"

Article Title: Immunogenicity and protective efficacy of recombinant alkaline shock protein 23 from Staphylococcus aureus in a murine model

Journal: Central-European Journal of Immunology

doi: 10.5114/ceji.2018.81348

Cloning, expression, purification and identification of Asp 23. The ~507 bp Asp 23 gene was cloned into pET 28a (+) vector, expressed in E. coli BL21 (DE3), purified by IMAC and the protein was identified using MALDI-TOF. A ) Isolation of Asp 23 gene by PCR. Lane 1, DNA marker; Lane 2, amplified asp23 . B ) Expression of asp23 in E. coli . Lane 1, supernatant from non-induced bacteria; Lanes 2-4, Supernatant from bacteria induced for expression of asp23 by addition of IPTG; Lane 5, Protein marker. C ) Asp23 after IMAC purification. Lane 1, Purified Asp23; Lane 2, Protein marker. D ) Spectrum obtained from MALDI-TOF analysis to determine the molecular mass of purified Asp23
Figure Legend Snippet: Cloning, expression, purification and identification of Asp 23. The ~507 bp Asp 23 gene was cloned into pET 28a (+) vector, expressed in E. coli BL21 (DE3), purified by IMAC and the protein was identified using MALDI-TOF. A ) Isolation of Asp 23 gene by PCR. Lane 1, DNA marker; Lane 2, amplified asp23 . B ) Expression of asp23 in E. coli . Lane 1, supernatant from non-induced bacteria; Lanes 2-4, Supernatant from bacteria induced for expression of asp23 by addition of IPTG; Lane 5, Protein marker. C ) Asp23 after IMAC purification. Lane 1, Purified Asp23; Lane 2, Protein marker. D ) Spectrum obtained from MALDI-TOF analysis to determine the molecular mass of purified Asp23

Techniques Used: Clone Assay, Expressing, Purification, Positron Emission Tomography, Plasmid Preparation, Isolation, Polymerase Chain Reaction, Marker, Amplification

6) Product Images from "Effect of blood type on anti-α-Gal immunity and the incidence of infectious diseases"

Article Title: Effect of blood type on anti-α-Gal immunity and the incidence of infectious diseases

Journal: Experimental & Molecular Medicine

doi: 10.1038/emm.2016.164

Correlation between anti-α-Gal antibodies and protection against malaria and tuberculosis. ( a ) Flow cytometry and immunofluorescence showing the presence of α-Gal on the surface of mycobacteria ( Mycobacterium marinum CECT 7091 reference strain). Escherichia coli O86:B7 (ATCC 12701) and BL21 (DE3) strains were included as positive and negative controls for α-Gal, respectively. For flow cytometry, cells were stained with BSI-IB 4 -FITC to visualize α-Gal and the viable cell population was gated according to forward scatter and side scatter parameters. Ten microliters of the fixed and stained samples were also used for immunofluorescence assays after air-drying and mounted in ProLong Antifade reagent containing DAPI. Representative immunofluorescence images are shown for bacterial cells stained with BSI-IB 4 -FITC (anti-α-Gal-FITC, green; blue, DAPI). ( b ) Anti-α-Gal antibody titers in patients with malaria or tuberculosis and healthy individuals. The levels of anti-α-Gal IgM and IgG antibodies were significantly higher in P. falciparum uninfected ( Pf− ) vs infected ( Pf +) individuals from Senegal. A similar pattern was observed in M. tuberculosis uninfected ( Mt −) vs infected ( Mt +) individuals from the Iberian Peninsula. The levels of anti-α-Gal IgE antibodies were lower in both Mt + and Pf + patients, when compared with Mt − and Pf − healthy individuals, respectively. The nonparametric Mann–Whitney U -test was used to compare values between infected and uninfected groups ( P =0.05).
Figure Legend Snippet: Correlation between anti-α-Gal antibodies and protection against malaria and tuberculosis. ( a ) Flow cytometry and immunofluorescence showing the presence of α-Gal on the surface of mycobacteria ( Mycobacterium marinum CECT 7091 reference strain). Escherichia coli O86:B7 (ATCC 12701) and BL21 (DE3) strains were included as positive and negative controls for α-Gal, respectively. For flow cytometry, cells were stained with BSI-IB 4 -FITC to visualize α-Gal and the viable cell population was gated according to forward scatter and side scatter parameters. Ten microliters of the fixed and stained samples were also used for immunofluorescence assays after air-drying and mounted in ProLong Antifade reagent containing DAPI. Representative immunofluorescence images are shown for bacterial cells stained with BSI-IB 4 -FITC (anti-α-Gal-FITC, green; blue, DAPI). ( b ) Anti-α-Gal antibody titers in patients with malaria or tuberculosis and healthy individuals. The levels of anti-α-Gal IgM and IgG antibodies were significantly higher in P. falciparum uninfected ( Pf− ) vs infected ( Pf +) individuals from Senegal. A similar pattern was observed in M. tuberculosis uninfected ( Mt −) vs infected ( Mt +) individuals from the Iberian Peninsula. The levels of anti-α-Gal IgE antibodies were lower in both Mt + and Pf + patients, when compared with Mt − and Pf − healthy individuals, respectively. The nonparametric Mann–Whitney U -test was used to compare values between infected and uninfected groups ( P =0.05).

Techniques Used: Flow Cytometry, Cytometry, Immunofluorescence, Staining, Infection, MANN-WHITNEY

7) Product Images from "Biological Role of Trichoderma harzianum-Derived Platelet-Activating Factor Acetylhydrolase (PAF-AH) on Stress Response and Antagonism"

Article Title: Biological Role of Trichoderma harzianum-Derived Platelet-Activating Factor Acetylhydrolase (PAF-AH) on Stress Response and Antagonism

Journal: PLoS ONE

doi: 10.1371/journal.pone.0100367

Properties of the PAF-AH enzyme. (A) The protein was expressed in E. coli BL21 (DE3). M, protein marker. 1, supernatant of induced lysis cells; 2 and 3, purified PAF-AH from the elution buffer. (B) Immunoblot analysis of purified PAF-AH using anti-His tag antibodies as indicated. (C) The PAF-AH activity assay experiment was carried out as described in Materials and Methods . Error bars denote SD. Asterisks indicate significant differences in enzyme activity under the condition of no Ca 2+ compared with 1 mM Ca 2+ (Student's t -test, P
Figure Legend Snippet: Properties of the PAF-AH enzyme. (A) The protein was expressed in E. coli BL21 (DE3). M, protein marker. 1, supernatant of induced lysis cells; 2 and 3, purified PAF-AH from the elution buffer. (B) Immunoblot analysis of purified PAF-AH using anti-His tag antibodies as indicated. (C) The PAF-AH activity assay experiment was carried out as described in Materials and Methods . Error bars denote SD. Asterisks indicate significant differences in enzyme activity under the condition of no Ca 2+ compared with 1 mM Ca 2+ (Student's t -test, P

Techniques Used: Marker, Lysis, Purification, Activity Assay

8) Product Images from "A Multiprotein DNA Translocation Complex Directs Intramycelial Plasmid Spreading during Streptomyces Conjugation"

Article Title: A Multiprotein DNA Translocation Complex Directs Intramycelial Plasmid Spreading during Streptomyces Conjugation

Journal: mBio

doi: 10.1128/mBio.02559-14

SpdB2-His promotes spheroplast formation by T7 lysozyme and forms unstable pores in planar lipid bilayers. (A) When expression of SpdB2-His was induced in BL21/pLysS, spheroplast (arrows) were formed. In contrast, spheroplasts were not observed in the absence of spdB2 (B) or when expressing spdB2 in BL21 lacking the T7 amidase (C). (D) When purified SpdB2-His was added to the cis side of a lipid bilayer, SpdB2-His inserted into the planar lipid bilayer, showing flickering pore structures. (E) The corresponding all-points conductance level histogram shows that there is no discrete conductance state of the open pore indicated by the long tail in the histogram.
Figure Legend Snippet: SpdB2-His promotes spheroplast formation by T7 lysozyme and forms unstable pores in planar lipid bilayers. (A) When expression of SpdB2-His was induced in BL21/pLysS, spheroplast (arrows) were formed. In contrast, spheroplasts were not observed in the absence of spdB2 (B) or when expressing spdB2 in BL21 lacking the T7 amidase (C). (D) When purified SpdB2-His was added to the cis side of a lipid bilayer, SpdB2-His inserted into the planar lipid bilayer, showing flickering pore structures. (E) The corresponding all-points conductance level histogram shows that there is no discrete conductance state of the open pore indicated by the long tail in the histogram.

Techniques Used: Expressing, Purification

9) Product Images from "Interactions of SARS Coronavirus Nucleocapsid Protein with the host cell proteasome subunit p42"

Article Title: Interactions of SARS Coronavirus Nucleocapsid Protein with the host cell proteasome subunit p42

Journal: Virology Journal

doi: 10.1186/1743-422X-7-99

Expression and Identification of SNP22b (SARS-CoV NP) . ( A ) Supernatants (lanes 1 and 10) and pellets (lanes 2 and 6) of E. coli BL21 containing the pET22b-SNP22b vector expressing SARS-CoV NP, without IPTG induction. Supernatants (lanes 3 and 7) and pellets (lanes 4 and 8) of E. coli BL21 containing the pET22b-SNP22b vector and expressing SARS-CoV NP, induced with IPTG. Pellets of E . coli BL21 containing pET22b induced with IPTG (lane 5). Molecular weight markers (lane 9). The position of SARS-CoV NP (~47 kDa) is indicated by an arrow. ( B ) Western blot analysis of SARS-CoV NP using an anti-SARS-CoV NP horse polyclonal antibody. Lysate of E . coli BL21 expressing SARS-CoV NP, induced by IPTG (lane 1). Lysate of E . coli BL21 containing pET22b induced by IPTG (lane 2). Molecular weight markers (lane 3). The position of SARS-CoV NP (~47 kDa) is indicated by an arrow.
Figure Legend Snippet: Expression and Identification of SNP22b (SARS-CoV NP) . ( A ) Supernatants (lanes 1 and 10) and pellets (lanes 2 and 6) of E. coli BL21 containing the pET22b-SNP22b vector expressing SARS-CoV NP, without IPTG induction. Supernatants (lanes 3 and 7) and pellets (lanes 4 and 8) of E. coli BL21 containing the pET22b-SNP22b vector and expressing SARS-CoV NP, induced with IPTG. Pellets of E . coli BL21 containing pET22b induced with IPTG (lane 5). Molecular weight markers (lane 9). The position of SARS-CoV NP (~47 kDa) is indicated by an arrow. ( B ) Western blot analysis of SARS-CoV NP using an anti-SARS-CoV NP horse polyclonal antibody. Lysate of E . coli BL21 expressing SARS-CoV NP, induced by IPTG (lane 1). Lysate of E . coli BL21 containing pET22b induced by IPTG (lane 2). Molecular weight markers (lane 3). The position of SARS-CoV NP (~47 kDa) is indicated by an arrow.

Techniques Used: Expressing, Plasmid Preparation, Molecular Weight, Western Blot

Expression and identification of GST and the p42-GST fusion protein . ( A ) Lysate of E . coli BL21 containing the pGEX-5X-1-p42 vector, expressing p42-GST fusion protein without IPTG induction (lanes 1, 5) or induced by IPTG (lanes 2, 4). Lysate of E . coli BL21 containing the pGEX-5X-1 vector expressing GST with IPTG induction (lane 6) or without IPTG (lane 7). Molecular weight markers (lane 3). ( B ) Western blot analysis of p42-GST fusion protein and GST expression using an anti-GST mAb. Lysates of GST-expressing E . coli BL21 with IPTG induction (lanes 1, 2). Lysates of p42-GST fusion protein-expressing E . coli BL21 with IPTG induction (lanes 6, 7). Supernatants of E . coli BL21 expressing GST lysed using lysozyme (lane 3). Supernatants of E . coli BL21 expressing p42-GST fusion protein lysed by lysozyme (lane 5). Molecular weight markers (lane 4).
Figure Legend Snippet: Expression and identification of GST and the p42-GST fusion protein . ( A ) Lysate of E . coli BL21 containing the pGEX-5X-1-p42 vector, expressing p42-GST fusion protein without IPTG induction (lanes 1, 5) or induced by IPTG (lanes 2, 4). Lysate of E . coli BL21 containing the pGEX-5X-1 vector expressing GST with IPTG induction (lane 6) or without IPTG (lane 7). Molecular weight markers (lane 3). ( B ) Western blot analysis of p42-GST fusion protein and GST expression using an anti-GST mAb. Lysates of GST-expressing E . coli BL21 with IPTG induction (lanes 1, 2). Lysates of p42-GST fusion protein-expressing E . coli BL21 with IPTG induction (lanes 6, 7). Supernatants of E . coli BL21 expressing GST lysed using lysozyme (lane 3). Supernatants of E . coli BL21 expressing p42-GST fusion protein lysed by lysozyme (lane 5). Molecular weight markers (lane 4).

Techniques Used: Expressing, Plasmid Preparation, Molecular Weight, Western Blot

10) Product Images from "Datasets of a novel bivalent single chain antibody constructed by overlapping oligonucleotide annealing method targeting human CD123"

Article Title: Datasets of a novel bivalent single chain antibody constructed by overlapping oligonucleotide annealing method targeting human CD123

Journal: Data in Brief

doi: 10.1016/j.dib.2016.07.014

Expression and purification of biscFv. A) SDS-PAGE of expression, Coomassie brilliant blue staining. Lane M1 and M2: PageRuler prestained protein ladder (Thermo fisher scientific, USA), Lane1: periplasmic extract of E.coli BL21 without pET22-biscFv plasmids (negative control), Lane2: periplasmic extract before passing into the HisTrap column and lane3: purified biscFv. B) Western blot of purified biscFv using anti-His monoclonal antibody and HRP conjugated goat-anti mouse. Lane M1 and M2: PageRuler prestained protein ladder, lane 1: periplasmic extract of E.coli BL21 without pET22-biscFv plasmid as negative control, lane 2: periplasmic extract before passing into the HisTrap column, and lane3: purified biscFv.
Figure Legend Snippet: Expression and purification of biscFv. A) SDS-PAGE of expression, Coomassie brilliant blue staining. Lane M1 and M2: PageRuler prestained protein ladder (Thermo fisher scientific, USA), Lane1: periplasmic extract of E.coli BL21 without pET22-biscFv plasmids (negative control), Lane2: periplasmic extract before passing into the HisTrap column and lane3: purified biscFv. B) Western blot of purified biscFv using anti-His monoclonal antibody and HRP conjugated goat-anti mouse. Lane M1 and M2: PageRuler prestained protein ladder, lane 1: periplasmic extract of E.coli BL21 without pET22-biscFv plasmid as negative control, lane 2: periplasmic extract before passing into the HisTrap column, and lane3: purified biscFv.

Techniques Used: Expressing, Purification, SDS Page, Staining, Negative Control, Western Blot, Plasmid Preparation

11) Product Images from "Development of New Recombinant DgK Antigen for Diagnosis of Dirofilaria immitis Infections in Dogs Using ELISA Technique and Its Comparison to Molecular Methods"

Article Title: Development of New Recombinant DgK Antigen for Diagnosis of Dirofilaria immitis Infections in Dogs Using ELISA Technique and Its Comparison to Molecular Methods

Journal: Iranian Biomedical Journal

doi: 10.22034/ibj.22.4.283

SDS-PAGE analysis of purified antigen and Western blot analysis of rDgK antigen. (a) Purified protein was loaded on 12% acrylamide gel, stained with Coomassie brilliant blue R-250. Lane M, protein molecular standard; lane 1, SDS-PAGE of purified recombinant protein (arrow). (b) Lane M, protein molecular standard; lane 1, Western blotting of expressed rDgK probed with (lane 1) the negative control pool sera, (lane 2) suspected dog pool sera, and (lane 3) the positive-control serum pools (arrows). Lane 4 indicates total protein of untransformed E. coli BL21 in Western blot with the positive-control serum pools.
Figure Legend Snippet: SDS-PAGE analysis of purified antigen and Western blot analysis of rDgK antigen. (a) Purified protein was loaded on 12% acrylamide gel, stained with Coomassie brilliant blue R-250. Lane M, protein molecular standard; lane 1, SDS-PAGE of purified recombinant protein (arrow). (b) Lane M, protein molecular standard; lane 1, Western blotting of expressed rDgK probed with (lane 1) the negative control pool sera, (lane 2) suspected dog pool sera, and (lane 3) the positive-control serum pools (arrows). Lane 4 indicates total protein of untransformed E. coli BL21 in Western blot with the positive-control serum pools.

Techniques Used: SDS Page, Purification, Western Blot, Acrylamide Gel Assay, Staining, Recombinant, Negative Control, Positive Control

Related Articles

Clone Assay:

Article Title: Interactions of SARS Coronavirus Nucleocapsid Protein with the host cell proteasome subunit p42
Article Snippet: All of the recombinant clones were confirmed by sequencing. .. Escherichia coli DH5α and BL21 (DE3) were obtained from Invitrogen (USA).

Article Title: A Bacterial Surface Display System Expressing Cleavable Capsid Proteins of Human Norovirus: A Novel System to Discover Candidate Receptors
Article Snippet: Bacterial Strains and Construction of the Recombinant Plasmids Competent E. coli DH5α and BL21 (Thermo Fisher, Shanghai, China) were used for recombinant plasmid construction and protein expression, respectively. .. Oligonucleotide “TB” coding for the peptide sequence of Leu-Val-Pro-Arg-Gly-Ser, was synthesized by Suzhou GENEWIZ Bio-Technology, Co., Ltd. Then, the artificially synthesized inaQn-TB sequence was inserted into cloning vector pUC57 to create pUC57-inaQn-TB.

Article Title: Production of Active Nonglycosylated Recombinant B-Chain of Type-2 Ribosome-Inactivating Protein from Viscum articulatum and Its Biological Effects on Peripheral Blood Mononuclear Cells
Article Snippet: Bacterial Strains and Plasmids Escherichia coli XL1 blue (F− φ 80(lacZ )ΔM15 ΔlacX74 hsdR (rk − , mk + ) ΔrecA1398 endA1 tonA ) and BL21 [F− ompT hsdSB (rB − mB − ) gal dcm (DE3) pLysS(CamR )] were obtained from Invitrogen. .. Plasmid pCR2.1-TOPO (Invitrogen) was used for cloning and sequencing, plasmid pET-28a(+) (Novagen) for expression.

Article Title: Development of New Recombinant DgK Antigen for Diagnosis of Dirofilaria immitis Infections in Dogs Using ELISA Technique and Its Comparison to Molecular Methods
Article Snippet: .. E. coli strains DH5α and BL21 (DE3) and the pTG19-T (Invitrogen, Barcelona, Spain) and pET-24a(+) (Biomatik, Canada) plasmids were used for cloning and recombinant protein expression, respectively. .. Bacterial cells were grown in LB agar and LB broth (Merck, Darmstadt, Germany) and incubated at 30°C overnight and 4 h, respectively.

Article Title: Development of New Recombinant DgK Antigen for Diagnosis of Dirofilaria immitis Infections in Dogs Using ELISA Technique and Its Comparison to Molecular Methods
Article Snippet: .. Plasmids, bacterial strains, and growth condition E. coli strains DH5α and BL21 (DE3) and the pTG19-T (Invitrogen, Barcelona, Spain) and pET-24a(+) (Biomatik, Canada) plasmids were used for cloning and recombinant protein expression, respectively. .. Bacterial cells were grown in LB agar and LB broth (Merck, Darmstadt, Germany) and incubated at 30°C overnight and 4 h, respectively.

Article Title: Immunogenicity and protective efficacy of recombinant alkaline shock protein 23 from Staphylococcus aureus in a murine model
Article Snippet: .. E. coli strains AB5α and BL21 (DE3) were routinely cultured on Luria Bertani (LB) agar or in broth at 37°C. pJET1.2 (Fermentas) and pET28a (+) (Novagen) were used as vectors for cloning and expression. .. After transformation with pJET, DH5α was grown in LB medium supplemented with 100 μg/ml of ampicillin.

Amplification:

Article Title: Interactions of SARS Coronavirus Nucleocapsid Protein with the host cell proteasome subunit p42
Article Snippet: The p42 gene was amplified by RT-PCR using mRNA from 2BS cells with the following primers: p42 forward: 5'-GATGAATTCATGGCGGACCCTAGAGATAAGG-3', reverse: 5'-GATCTCGAGTTACACAGGTTTGTAGTCCAATTTAG-3'. .. Escherichia coli DH5α and BL21 (DE3) were obtained from Invitrogen (USA).

Article Title: Enzymatic Conversion of RBCs by α-N-Acetylgalactosaminidase from Spirosoma linguale
Article Snippet: The pCR®-Blunt vector and the competent cells E. coli DH5α and BL21 (DE3) and gel chromatography column were obtained from Invitrogen, Carlsbad, California. .. Gene amplification was completed using an Eppendorf Mastercycler® pro thermal cycler, Hauppauge, New York.

Filtration:

Article Title: Enzymatic Conversion of RBCs by α-N-Acetylgalactosaminidase from Spirosoma linguale
Article Snippet: Glycosidase conjugates and gel filtration molecular weight markers were obtained from Sigma-Aldrich Company, St. Louis, Missouri. .. The pCR®-Blunt vector and the competent cells E. coli DH5α and BL21 (DE3) and gel chromatography column were obtained from Invitrogen, Carlsbad, California.

Synthesized:

Article Title: A Bacterial Surface Display System Expressing Cleavable Capsid Proteins of Human Norovirus: A Novel System to Discover Candidate Receptors
Article Snippet: Bacterial Strains and Construction of the Recombinant Plasmids Competent E. coli DH5α and BL21 (Thermo Fisher, Shanghai, China) were used for recombinant plasmid construction and protein expression, respectively. .. Oligonucleotide “TB” coding for the peptide sequence of Leu-Val-Pro-Arg-Gly-Ser, was synthesized by Suzhou GENEWIZ Bio-Technology, Co., Ltd. Then, the artificially synthesized inaQn-TB sequence was inserted into cloning vector pUC57 to create pUC57-inaQn-TB.

Construct:

Article Title: Interactions of SARS Coronavirus Nucleocapsid Protein with the host cell proteasome subunit p42
Article Snippet: The plasmid pcDNA3.0-SNP22b was constructed by subcloning the SNP gene, released from pET22b-SNP22b by BamHI/EcoRI, into pcDNA3.0 (Invitrogen). .. Escherichia coli DH5α and BL21 (DE3) were obtained from Invitrogen (USA).

Article Title: A Bacterial Surface Display System Expressing Cleavable Capsid Proteins of Human Norovirus: A Novel System to Discover Candidate Receptors
Article Snippet: Bacterial Strains and Construction of the Recombinant Plasmids Competent E. coli DH5α and BL21 (Thermo Fisher, Shanghai, China) were used for recombinant plasmid construction and protein expression, respectively. .. Similarly, pET28a-inaQn-TB was constructed from pET28a-inaQn for use as a negative control.

Electrophoresis:

Article Title: Datasets of a novel bivalent single chain antibody constructed by overlapping oligonucleotide annealing method targeting human CD123
Article Snippet: BL21 and purification by immobilized metal affinity chromatography (IMAC), the Purified biscFvs were analyzed by SDS-PAGE and western blotting using prestained Protein Page Ruler (Thermo Fisher Scientific, USA) as described below. .. After electrophoresis, the protein bands were visualized by Coomassie Brilliant Blue staining or were electrophoretically transferred to a nitrocellulose membrane and blocked for 16 h at 4 °C using 3% BSA in Tris-buffered saline (TBS), followed by four washes of 5 min in 20 ml of TBS, 0.1% (v/v) Tween 20.

Incubation:

Article Title: Development of New Recombinant DgK Antigen for Diagnosis of Dirofilaria immitis Infections in Dogs Using ELISA Technique and Its Comparison to Molecular Methods
Article Snippet: E. coli strains DH5α and BL21 (DE3) and the pTG19-T (Invitrogen, Barcelona, Spain) and pET-24a(+) (Biomatik, Canada) plasmids were used for cloning and recombinant protein expression, respectively. .. Bacterial cells were grown in LB agar and LB broth (Merck, Darmstadt, Germany) and incubated at 30°C overnight and 4 h, respectively.

Article Title: Effect of blood type on anti-α-Gal immunity and the incidence of infectious diseases
Article Snippet: .. The Escherichia coli O86:B7 (ATCC 12701) and BL21 (DE3; Invitrogen, Carlsbad, CA, USA) strains were included as positive and negative controls for α-Gal, respectively, inoculated into 50 ml of Luria Broth (LB), and incubated at 37 °C overnight. ..

Article Title: Development of New Recombinant DgK Antigen for Diagnosis of Dirofilaria immitis Infections in Dogs Using ELISA Technique and Its Comparison to Molecular Methods
Article Snippet: Plasmids, bacterial strains, and growth condition E. coli strains DH5α and BL21 (DE3) and the pTG19-T (Invitrogen, Barcelona, Spain) and pET-24a(+) (Biomatik, Canada) plasmids were used for cloning and recombinant protein expression, respectively. .. Bacterial cells were grown in LB agar and LB broth (Merck, Darmstadt, Germany) and incubated at 30°C overnight and 4 h, respectively.

Expressing:

Article Title: A Multiprotein DNA Translocation Complex Directs Intramycelial Plasmid Spreading during Streptomyces Conjugation
Article Snippet: Proteins were purified from E. coli Rosetta 2 (Merck), BL21(DE3), or BL21(DE3)/pLysS (Invitrogen) and S. lividans TK23 ( ). .. Oligonucleotides used for PCR targeting, two-hybrid analyses, gene expression, and DNA binding studies are given in .

Article Title: Datasets of a novel bivalent single chain antibody constructed by overlapping oligonucleotide annealing method targeting human CD123
Article Snippet: 3.1 SDS-PAGE, Western blotting Following expression in Ecoli. .. BL21 and purification by immobilized metal affinity chromatography (IMAC), the Purified biscFvs were analyzed by SDS-PAGE and western blotting using prestained Protein Page Ruler (Thermo Fisher Scientific, USA) as described below.

Article Title: A Bacterial Surface Display System Expressing Cleavable Capsid Proteins of Human Norovirus: A Novel System to Discover Candidate Receptors
Article Snippet: .. Bacterial Strains and Construction of the Recombinant Plasmids Competent E. coli DH5α and BL21 (Thermo Fisher, Shanghai, China) were used for recombinant plasmid construction and protein expression, respectively. .. Oligonucleotide “TB” coding for the peptide sequence of Leu-Val-Pro-Arg-Gly-Ser, was synthesized by Suzhou GENEWIZ Bio-Technology, Co., Ltd. Then, the artificially synthesized inaQn-TB sequence was inserted into cloning vector pUC57 to create pUC57-inaQn-TB.

Article Title: Development of New Recombinant DgK Antigen for Diagnosis of Dirofilaria immitis Infections in Dogs Using ELISA Technique and Its Comparison to Molecular Methods
Article Snippet: .. E. coli strains DH5α and BL21 (DE3) and the pTG19-T (Invitrogen, Barcelona, Spain) and pET-24a(+) (Biomatik, Canada) plasmids were used for cloning and recombinant protein expression, respectively. .. Bacterial cells were grown in LB agar and LB broth (Merck, Darmstadt, Germany) and incubated at 30°C overnight and 4 h, respectively.

Article Title: Development of New Recombinant DgK Antigen for Diagnosis of Dirofilaria immitis Infections in Dogs Using ELISA Technique and Its Comparison to Molecular Methods
Article Snippet: .. Plasmids, bacterial strains, and growth condition E. coli strains DH5α and BL21 (DE3) and the pTG19-T (Invitrogen, Barcelona, Spain) and pET-24a(+) (Biomatik, Canada) plasmids were used for cloning and recombinant protein expression, respectively. .. Bacterial cells were grown in LB agar and LB broth (Merck, Darmstadt, Germany) and incubated at 30°C overnight and 4 h, respectively.

Article Title: Expression and characterization of highly antigenic domains of chicken anemia virus viral VP2 and VP3 subunit proteins in a recombinant E. coli for sero-diagnostic applications
Article Snippet: Bacterial strains and inoculation Two E. coli strains, BL21 (DE3) and BL21(DE3)-pLysS were purchased from Invitrogen Life Technologies (Carlsbad, CA) and Stratagene (La Jolla, CA), respectively. .. The overnight culture were then inoculated into 50 ml LB medium and grown at 37°C for 3 h by which time the optical density of the culture had reach 0.5 of OD600 and could be used for competent cell preparation and protein expression.

Article Title: Biological Role of Trichoderma harzianum-Derived Platelet-Activating Factor Acetylhydrolase (PAF-AH) on Stress Response and Antagonism
Article Snippet: .. Escherichia coli DH5α and BL21 (DE3) were purchased from Invitrogen (Carlsbad, CA, USA) for plasmid propagation and protein expression, respectively. ..

Article Title: Immunogenicity and protective efficacy of recombinant alkaline shock protein 23 from Staphylococcus aureus in a murine model
Article Snippet: .. E. coli strains AB5α and BL21 (DE3) were routinely cultured on Luria Bertani (LB) agar or in broth at 37°C. pJET1.2 (Fermentas) and pET28a (+) (Novagen) were used as vectors for cloning and expression. .. After transformation with pJET, DH5α was grown in LB medium supplemented with 100 μg/ml of ampicillin.

BIA-KA:

Article Title: Enzymatic Conversion of RBCs by α-N-Acetylgalactosaminidase from Spirosoma linguale
Article Snippet: Chromatography columns (HisTrap, Q Sepharose, and Zorbax GF-250), SDS-PAGE materials (gels, buffers, and protein markers), QuickChange® Site-Directed Mutagenesis Kit (Stratagene), Western Blot materials (Nitrocellulose Bio Trace™ Pall Life Science, buffers, and developing reagent), BCA and Bradford protein determination solutions, BSA standards, restriction enzymes, the competent cells E. coli CD41 (DE3), and TEV protease were obtained from Fisher Scientific, Waltham, Massachusetts. .. The pCR®-Blunt vector and the competent cells E. coli DH5α and BL21 (DE3) and gel chromatography column were obtained from Invitrogen, Carlsbad, California.

Western Blot:

Article Title: Datasets of a novel bivalent single chain antibody constructed by overlapping oligonucleotide annealing method targeting human CD123
Article Snippet: .. BL21 and purification by immobilized metal affinity chromatography (IMAC), the Purified biscFvs were analyzed by SDS-PAGE and western blotting using prestained Protein Page Ruler (Thermo Fisher Scientific, USA) as described below. .. After electrophoresis, the protein bands were visualized by Coomassie Brilliant Blue staining or were electrophoretically transferred to a nitrocellulose membrane and blocked for 16 h at 4 °C using 3% BSA in Tris-buffered saline (TBS), followed by four washes of 5 min in 20 ml of TBS, 0.1% (v/v) Tween 20.

Article Title: Enzymatic Conversion of RBCs by α-N-Acetylgalactosaminidase from Spirosoma linguale
Article Snippet: Chromatography columns (HisTrap, Q Sepharose, and Zorbax GF-250), SDS-PAGE materials (gels, buffers, and protein markers), QuickChange® Site-Directed Mutagenesis Kit (Stratagene), Western Blot materials (Nitrocellulose Bio Trace™ Pall Life Science, buffers, and developing reagent), BCA and Bradford protein determination solutions, BSA standards, restriction enzymes, the competent cells E. coli CD41 (DE3), and TEV protease were obtained from Fisher Scientific, Waltham, Massachusetts. .. The pCR®-Blunt vector and the competent cells E. coli DH5α and BL21 (DE3) and gel chromatography column were obtained from Invitrogen, Carlsbad, California.

Transformation Assay:

Article Title: Immunogenicity and protective efficacy of recombinant alkaline shock protein 23 from Staphylococcus aureus in a murine model
Article Snippet: E. coli strains AB5α and BL21 (DE3) were routinely cultured on Luria Bertani (LB) agar or in broth at 37°C. pJET1.2 (Fermentas) and pET28a (+) (Novagen) were used as vectors for cloning and expression. .. After transformation with pJET, DH5α was grown in LB medium supplemented with 100 μg/ml of ampicillin.

Chromatography:

Article Title: Enzymatic Conversion of RBCs by α-N-Acetylgalactosaminidase from Spirosoma linguale
Article Snippet: .. The pCR®-Blunt vector and the competent cells E. coli DH5α and BL21 (DE3) and gel chromatography column were obtained from Invitrogen, Carlsbad, California. .. The pKA8H vector was obtained from the University of Missouri.

Cell Culture:

Article Title: Immunogenicity and protective efficacy of recombinant alkaline shock protein 23 from Staphylococcus aureus in a murine model
Article Snippet: .. E. coli strains AB5α and BL21 (DE3) were routinely cultured on Luria Bertani (LB) agar or in broth at 37°C. pJET1.2 (Fermentas) and pET28a (+) (Novagen) were used as vectors for cloning and expression. .. After transformation with pJET, DH5α was grown in LB medium supplemented with 100 μg/ml of ampicillin.

Polymerase Chain Reaction:

Article Title: A Multiprotein DNA Translocation Complex Directs Intramycelial Plasmid Spreading during Streptomyces Conjugation
Article Snippet: Proteins were purified from E. coli Rosetta 2 (Merck), BL21(DE3), or BL21(DE3)/pLysS (Invitrogen) and S. lividans TK23 ( ). .. Oligonucleotides used for PCR targeting, two-hybrid analyses, gene expression, and DNA binding studies are given in .

Article Title: Interactions of SARS Coronavirus Nucleocapsid Protein with the host cell proteasome subunit p42
Article Snippet: PCR products were purified and cloned into the pGEX-5X-1 plasmid (Pharmacia, GE) using XhoI/EcoRI, to generate pGEX-5X-1-p42. .. Escherichia coli DH5α and BL21 (DE3) were obtained from Invitrogen (USA).

Article Title: Enzymatic Conversion of RBCs by α-N-Acetylgalactosaminidase from Spirosoma linguale
Article Snippet: .. The pCR®-Blunt vector and the competent cells E. coli DH5α and BL21 (DE3) and gel chromatography column were obtained from Invitrogen, Carlsbad, California. .. The pKA8H vector was obtained from the University of Missouri.

Sequencing:

Article Title: Interactions of SARS Coronavirus Nucleocapsid Protein with the host cell proteasome subunit p42
Article Snippet: All of the recombinant clones were confirmed by sequencing. .. Escherichia coli DH5α and BL21 (DE3) were obtained from Invitrogen (USA).

Article Title: A Bacterial Surface Display System Expressing Cleavable Capsid Proteins of Human Norovirus: A Novel System to Discover Candidate Receptors
Article Snippet: Bacterial Strains and Construction of the Recombinant Plasmids Competent E. coli DH5α and BL21 (Thermo Fisher, Shanghai, China) were used for recombinant plasmid construction and protein expression, respectively. .. Oligonucleotide “TB” coding for the peptide sequence of Leu-Val-Pro-Arg-Gly-Ser, was synthesized by Suzhou GENEWIZ Bio-Technology, Co., Ltd. Then, the artificially synthesized inaQn-TB sequence was inserted into cloning vector pUC57 to create pUC57-inaQn-TB.

Article Title: Production of Active Nonglycosylated Recombinant B-Chain of Type-2 Ribosome-Inactivating Protein from Viscum articulatum and Its Biological Effects on Peripheral Blood Mononuclear Cells
Article Snippet: Bacterial Strains and Plasmids Escherichia coli XL1 blue (F− φ 80(lacZ )ΔM15 ΔlacX74 hsdR (rk − , mk + ) ΔrecA1398 endA1 tonA ) and BL21 [F− ompT hsdSB (rB − mB − ) gal dcm (DE3) pLysS(CamR )] were obtained from Invitrogen. .. Plasmid pCR2.1-TOPO (Invitrogen) was used for cloning and sequencing, plasmid pET-28a(+) (Novagen) for expression.

Recombinant:

Article Title: Interactions of SARS Coronavirus Nucleocapsid Protein with the host cell proteasome subunit p42
Article Snippet: All of the recombinant clones were confirmed by sequencing. .. Escherichia coli DH5α and BL21 (DE3) were obtained from Invitrogen (USA).

Article Title: A Bacterial Surface Display System Expressing Cleavable Capsid Proteins of Human Norovirus: A Novel System to Discover Candidate Receptors
Article Snippet: .. Bacterial Strains and Construction of the Recombinant Plasmids Competent E. coli DH5α and BL21 (Thermo Fisher, Shanghai, China) were used for recombinant plasmid construction and protein expression, respectively. .. Oligonucleotide “TB” coding for the peptide sequence of Leu-Val-Pro-Arg-Gly-Ser, was synthesized by Suzhou GENEWIZ Bio-Technology, Co., Ltd. Then, the artificially synthesized inaQn-TB sequence was inserted into cloning vector pUC57 to create pUC57-inaQn-TB.

Article Title: Development of New Recombinant DgK Antigen for Diagnosis of Dirofilaria immitis Infections in Dogs Using ELISA Technique and Its Comparison to Molecular Methods
Article Snippet: .. E. coli strains DH5α and BL21 (DE3) and the pTG19-T (Invitrogen, Barcelona, Spain) and pET-24a(+) (Biomatik, Canada) plasmids were used for cloning and recombinant protein expression, respectively. .. Bacterial cells were grown in LB agar and LB broth (Merck, Darmstadt, Germany) and incubated at 30°C overnight and 4 h, respectively.

Article Title: Development of New Recombinant DgK Antigen for Diagnosis of Dirofilaria immitis Infections in Dogs Using ELISA Technique and Its Comparison to Molecular Methods
Article Snippet: .. Plasmids, bacterial strains, and growth condition E. coli strains DH5α and BL21 (DE3) and the pTG19-T (Invitrogen, Barcelona, Spain) and pET-24a(+) (Biomatik, Canada) plasmids were used for cloning and recombinant protein expression, respectively. .. Bacterial cells were grown in LB agar and LB broth (Merck, Darmstadt, Germany) and incubated at 30°C overnight and 4 h, respectively.

Molecular Weight:

Article Title: Enzymatic Conversion of RBCs by α-N-Acetylgalactosaminidase from Spirosoma linguale
Article Snippet: Glycosidase conjugates and gel filtration molecular weight markers were obtained from Sigma-Aldrich Company, St. Louis, Missouri. .. The pCR®-Blunt vector and the competent cells E. coli DH5α and BL21 (DE3) and gel chromatography column were obtained from Invitrogen, Carlsbad, California.

Mutagenesis:

Article Title: Enzymatic Conversion of RBCs by α-N-Acetylgalactosaminidase from Spirosoma linguale
Article Snippet: Chromatography columns (HisTrap, Q Sepharose, and Zorbax GF-250), SDS-PAGE materials (gels, buffers, and protein markers), QuickChange® Site-Directed Mutagenesis Kit (Stratagene), Western Blot materials (Nitrocellulose Bio Trace™ Pall Life Science, buffers, and developing reagent), BCA and Bradford protein determination solutions, BSA standards, restriction enzymes, the competent cells E. coli CD41 (DE3), and TEV protease were obtained from Fisher Scientific, Waltham, Massachusetts. .. The pCR®-Blunt vector and the competent cells E. coli DH5α and BL21 (DE3) and gel chromatography column were obtained from Invitrogen, Carlsbad, California.

Subcloning:

Article Title: Interactions of SARS Coronavirus Nucleocapsid Protein with the host cell proteasome subunit p42
Article Snippet: The plasmid pcDNA3.0-SNP22b was constructed by subcloning the SNP gene, released from pET22b-SNP22b by BamHI/EcoRI, into pcDNA3.0 (Invitrogen). .. Escherichia coli DH5α and BL21 (DE3) were obtained from Invitrogen (USA).

Purification:

Article Title: A Multiprotein DNA Translocation Complex Directs Intramycelial Plasmid Spreading during Streptomyces Conjugation
Article Snippet: .. Proteins were purified from E. coli Rosetta 2 (Merck), BL21(DE3), or BL21(DE3)/pLysS (Invitrogen) and S. lividans TK23 ( ). ..

Article Title: Datasets of a novel bivalent single chain antibody constructed by overlapping oligonucleotide annealing method targeting human CD123
Article Snippet: .. BL21 and purification by immobilized metal affinity chromatography (IMAC), the Purified biscFvs were analyzed by SDS-PAGE and western blotting using prestained Protein Page Ruler (Thermo Fisher Scientific, USA) as described below. .. After electrophoresis, the protein bands were visualized by Coomassie Brilliant Blue staining or were electrophoretically transferred to a nitrocellulose membrane and blocked for 16 h at 4 °C using 3% BSA in Tris-buffered saline (TBS), followed by four washes of 5 min in 20 ml of TBS, 0.1% (v/v) Tween 20.

Article Title: Interactions of SARS Coronavirus Nucleocapsid Protein with the host cell proteasome subunit p42
Article Snippet: PCR products were purified and cloned into the pGEX-5X-1 plasmid (Pharmacia, GE) using XhoI/EcoRI, to generate pGEX-5X-1-p42. .. Escherichia coli DH5α and BL21 (DE3) were obtained from Invitrogen (USA).

Protein Purification:

Article Title: Enzymatic Conversion of RBCs by α-N-Acetylgalactosaminidase from Spirosoma linguale
Article Snippet: The pCR®-Blunt vector and the competent cells E. coli DH5α and BL21 (DE3) and gel chromatography column were obtained from Invitrogen, Carlsbad, California. .. Protein purification was accomplished with an ÄKTAprime™ plus chromatography system from GE Healthcare, Piscataway, New Jersey.

Reverse Transcription Polymerase Chain Reaction:

Article Title: Interactions of SARS Coronavirus Nucleocapsid Protein with the host cell proteasome subunit p42
Article Snippet: The p42 gene was amplified by RT-PCR using mRNA from 2BS cells with the following primers: p42 forward: 5'-GATGAATTCATGGCGGACCCTAGAGATAAGG-3', reverse: 5'-GATCTCGAGTTACACAGGTTTGTAGTCCAATTTAG-3'. .. Escherichia coli DH5α and BL21 (DE3) were obtained from Invitrogen (USA).

Positron Emission Tomography:

Article Title: A Bacterial Surface Display System Expressing Cleavable Capsid Proteins of Human Norovirus: A Novel System to Discover Candidate Receptors
Article Snippet: Bacterial Strains and Construction of the Recombinant Plasmids Competent E. coli DH5α and BL21 (Thermo Fisher, Shanghai, China) were used for recombinant plasmid construction and protein expression, respectively. .. After re-digestion of plasmids pET28a, pET28a-P (GII.4) and pUC57-inaQn-TB ( Table ), the inaQn -TB and P (GII.4) fragment were inserted into pET-28a to create recombinant plasmid pET28a-inaQn-TB-P (GII.4).

Article Title: Production of Active Nonglycosylated Recombinant B-Chain of Type-2 Ribosome-Inactivating Protein from Viscum articulatum and Its Biological Effects on Peripheral Blood Mononuclear Cells
Article Snippet: Bacterial Strains and Plasmids Escherichia coli XL1 blue (F− φ 80(lacZ )ΔM15 ΔlacX74 hsdR (rk − , mk + ) ΔrecA1398 endA1 tonA ) and BL21 [F− ompT hsdSB (rB − mB − ) gal dcm (DE3) pLysS(CamR )] were obtained from Invitrogen. .. Plasmid pCR2.1-TOPO (Invitrogen) was used for cloning and sequencing, plasmid pET-28a(+) (Novagen) for expression.

Article Title: Development of New Recombinant DgK Antigen for Diagnosis of Dirofilaria immitis Infections in Dogs Using ELISA Technique and Its Comparison to Molecular Methods
Article Snippet: .. E. coli strains DH5α and BL21 (DE3) and the pTG19-T (Invitrogen, Barcelona, Spain) and pET-24a(+) (Biomatik, Canada) plasmids were used for cloning and recombinant protein expression, respectively. .. Bacterial cells were grown in LB agar and LB broth (Merck, Darmstadt, Germany) and incubated at 30°C overnight and 4 h, respectively.

Article Title: Development of New Recombinant DgK Antigen for Diagnosis of Dirofilaria immitis Infections in Dogs Using ELISA Technique and Its Comparison to Molecular Methods
Article Snippet: .. Plasmids, bacterial strains, and growth condition E. coli strains DH5α and BL21 (DE3) and the pTG19-T (Invitrogen, Barcelona, Spain) and pET-24a(+) (Biomatik, Canada) plasmids were used for cloning and recombinant protein expression, respectively. .. Bacterial cells were grown in LB agar and LB broth (Merck, Darmstadt, Germany) and incubated at 30°C overnight and 4 h, respectively.

Polyacrylamide Gel Electrophoresis:

Article Title: Datasets of a novel bivalent single chain antibody constructed by overlapping oligonucleotide annealing method targeting human CD123
Article Snippet: .. BL21 and purification by immobilized metal affinity chromatography (IMAC), the Purified biscFvs were analyzed by SDS-PAGE and western blotting using prestained Protein Page Ruler (Thermo Fisher Scientific, USA) as described below. .. After electrophoresis, the protein bands were visualized by Coomassie Brilliant Blue staining or were electrophoretically transferred to a nitrocellulose membrane and blocked for 16 h at 4 °C using 3% BSA in Tris-buffered saline (TBS), followed by four washes of 5 min in 20 ml of TBS, 0.1% (v/v) Tween 20.

SDS Page:

Article Title: Datasets of a novel bivalent single chain antibody constructed by overlapping oligonucleotide annealing method targeting human CD123
Article Snippet: .. BL21 and purification by immobilized metal affinity chromatography (IMAC), the Purified biscFvs were analyzed by SDS-PAGE and western blotting using prestained Protein Page Ruler (Thermo Fisher Scientific, USA) as described below. .. After electrophoresis, the protein bands were visualized by Coomassie Brilliant Blue staining or were electrophoretically transferred to a nitrocellulose membrane and blocked for 16 h at 4 °C using 3% BSA in Tris-buffered saline (TBS), followed by four washes of 5 min in 20 ml of TBS, 0.1% (v/v) Tween 20.

Article Title: Enzymatic Conversion of RBCs by α-N-Acetylgalactosaminidase from Spirosoma linguale
Article Snippet: Chromatography columns (HisTrap, Q Sepharose, and Zorbax GF-250), SDS-PAGE materials (gels, buffers, and protein markers), QuickChange® Site-Directed Mutagenesis Kit (Stratagene), Western Blot materials (Nitrocellulose Bio Trace™ Pall Life Science, buffers, and developing reagent), BCA and Bradford protein determination solutions, BSA standards, restriction enzymes, the competent cells E. coli CD41 (DE3), and TEV protease were obtained from Fisher Scientific, Waltham, Massachusetts. .. The pCR®-Blunt vector and the competent cells E. coli DH5α and BL21 (DE3) and gel chromatography column were obtained from Invitrogen, Carlsbad, California.

Plasmid Preparation:

Article Title: Interactions of SARS Coronavirus Nucleocapsid Protein with the host cell proteasome subunit p42
Article Snippet: The plasmid pcDNA3.0-SNP22b was constructed by subcloning the SNP gene, released from pET22b-SNP22b by BamHI/EcoRI, into pcDNA3.0 (Invitrogen). .. Escherichia coli DH5α and BL21 (DE3) were obtained from Invitrogen (USA).

Article Title: A Bacterial Surface Display System Expressing Cleavable Capsid Proteins of Human Norovirus: A Novel System to Discover Candidate Receptors
Article Snippet: .. Bacterial Strains and Construction of the Recombinant Plasmids Competent E. coli DH5α and BL21 (Thermo Fisher, Shanghai, China) were used for recombinant plasmid construction and protein expression, respectively. .. Oligonucleotide “TB” coding for the peptide sequence of Leu-Val-Pro-Arg-Gly-Ser, was synthesized by Suzhou GENEWIZ Bio-Technology, Co., Ltd. Then, the artificially synthesized inaQn-TB sequence was inserted into cloning vector pUC57 to create pUC57-inaQn-TB.

Article Title: Production of Active Nonglycosylated Recombinant B-Chain of Type-2 Ribosome-Inactivating Protein from Viscum articulatum and Its Biological Effects on Peripheral Blood Mononuclear Cells
Article Snippet: Bacterial Strains and Plasmids Escherichia coli XL1 blue (F− φ 80(lacZ )ΔM15 ΔlacX74 hsdR (rk − , mk + ) ΔrecA1398 endA1 tonA ) and BL21 [F− ompT hsdSB (rB − mB − ) gal dcm (DE3) pLysS(CamR )] were obtained from Invitrogen. .. Plasmid pCR2.1-TOPO (Invitrogen) was used for cloning and sequencing, plasmid pET-28a(+) (Novagen) for expression.

Article Title: Enzymatic Conversion of RBCs by α-N-Acetylgalactosaminidase from Spirosoma linguale
Article Snippet: .. The pCR®-Blunt vector and the competent cells E. coli DH5α and BL21 (DE3) and gel chromatography column were obtained from Invitrogen, Carlsbad, California. .. The pKA8H vector was obtained from the University of Missouri.

Article Title: Biological Role of Trichoderma harzianum-Derived Platelet-Activating Factor Acetylhydrolase (PAF-AH) on Stress Response and Antagonism
Article Snippet: .. Escherichia coli DH5α and BL21 (DE3) were purchased from Invitrogen (Carlsbad, CA, USA) for plasmid propagation and protein expression, respectively. ..

Negative Control:

Article Title: A Bacterial Surface Display System Expressing Cleavable Capsid Proteins of Human Norovirus: A Novel System to Discover Candidate Receptors
Article Snippet: Bacterial Strains and Construction of the Recombinant Plasmids Competent E. coli DH5α and BL21 (Thermo Fisher, Shanghai, China) were used for recombinant plasmid construction and protein expression, respectively. .. Similarly, pET28a-inaQn-TB was constructed from pET28a-inaQn for use as a negative control.

Binding Assay:

Article Title: A Multiprotein DNA Translocation Complex Directs Intramycelial Plasmid Spreading during Streptomyces Conjugation
Article Snippet: Proteins were purified from E. coli Rosetta 2 (Merck), BL21(DE3), or BL21(DE3)/pLysS (Invitrogen) and S. lividans TK23 ( ). .. Oligonucleotides used for PCR targeting, two-hybrid analyses, gene expression, and DNA binding studies are given in .

Affinity Chromatography:

Article Title: Datasets of a novel bivalent single chain antibody constructed by overlapping oligonucleotide annealing method targeting human CD123
Article Snippet: .. BL21 and purification by immobilized metal affinity chromatography (IMAC), the Purified biscFvs were analyzed by SDS-PAGE and western blotting using prestained Protein Page Ruler (Thermo Fisher Scientific, USA) as described below. .. After electrophoresis, the protein bands were visualized by Coomassie Brilliant Blue staining or were electrophoretically transferred to a nitrocellulose membrane and blocked for 16 h at 4 °C using 3% BSA in Tris-buffered saline (TBS), followed by four washes of 5 min in 20 ml of TBS, 0.1% (v/v) Tween 20.

Concentration Assay:

Article Title: Effect of blood type on anti-α-Gal immunity and the incidence of infectious diseases
Article Snippet: Spectrophotometric absorbance was measured at 600 nm (OD600 ) and the concentration was adjusted to 108 –109 CFU ml−1 . .. The Escherichia coli O86:B7 (ATCC 12701) and BL21 (DE3; Invitrogen, Carlsbad, CA, USA) strains were included as positive and negative controls for α-Gal, respectively, inoculated into 50 ml of Luria Broth (LB), and incubated at 37 °C overnight.

Staining:

Article Title: Datasets of a novel bivalent single chain antibody constructed by overlapping oligonucleotide annealing method targeting human CD123
Article Snippet: BL21 and purification by immobilized metal affinity chromatography (IMAC), the Purified biscFvs were analyzed by SDS-PAGE and western blotting using prestained Protein Page Ruler (Thermo Fisher Scientific, USA) as described below. .. After electrophoresis, the protein bands were visualized by Coomassie Brilliant Blue staining or were electrophoretically transferred to a nitrocellulose membrane and blocked for 16 h at 4 °C using 3% BSA in Tris-buffered saline (TBS), followed by four washes of 5 min in 20 ml of TBS, 0.1% (v/v) Tween 20.

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    Thermo Fisher e coli bl21
    KHSRP is modified by SUMO1 at the major site K87 in vitro and in cells. a-b Exogenous and endogenous KHSRP in cells are modified by SUMO1. 293T cells transfected with indicated plasmids were lysed and pulled down with Ni 2+ -NTA resin for SUMOylation assay, and SUMO1 modification of KHSRP was analyzed by Western blotting with indicated antibodies. c SUMO1 modification of KHSRP is verified by in vitro E.coli -based SUMOylation assay. Plasmid pGEX-4T-1-KHSRP was co-transformed with or without pE1E2SUMO1 plasmid into E.coli <t>BL21</t> (DE3). After GST pull-down purification, Western blotting was conducted with anti-SUMO1 antibody and the same membrane was detected with anti-GST antibody after stripping. d Mutation of K87R weakens SUMO1 modification of KHSRP in 293T cells. The construct pEF-5HA-KHSRP-WT, or -K87R, or -K359R, or -K628R was co-transfected with His-SUMO1 into 293T cells. 48 h after transfection, cells were lysed for the SUMOylation assay with Ni 2+ -NTA resin
    E Coli Bl21, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/e coli bl21/product/Thermo Fisher
    Average 99 stars, based on 10 article reviews
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    e coli bl21 - by Bioz Stars, 2020-04
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    Thermo Fisher plasmid interference assay bl21 ai
    Transcription-dependent loss of the target plasmid. <t>BL21-AI</t> strain with the STH Csm module plasmid was challenged with plasmids with STH target sequence or without a target sequence with or without IPTG (1μM) as indicated. (A) Plasmid targeting assay was performed as in Fig 2 (without arabinose). Corresponding Csm modules and targets are indicated with green boxes. (B) Presence of plasmids was assessed by plasmid extraction following overnight growth in medium with or without 1mM IPTG as indicated. Plasmid DNA was linearized, separated by agarose gel electrophoresis and visualized with ethidium bromide. Plasmid DNA from 4 individual colonies is shown for each condition. Positions of linearized Csm module and target plasmids are indicated. Dashed lines indicate rearranged non-contiguous sections of single original gel.
    Plasmid Interference Assay Bl21 Ai, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/plasmid interference assay bl21 ai/product/Thermo Fisher
    Average 92 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    plasmid interference assay bl21 ai - by Bioz Stars, 2020-04
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    92
    Thermo Fisher e coli bl21 cells
    Detection of recombinant plasmids and expression and purification of recombinant proteins M1: DNA marker (DL2000); lane 1: PCR products of candidate antigen genes; lane 2: enzyme digestion of recombinant plasmids with Bam HI + Xho I; lane 3: enzyme digestion of recombinant plasmids with Bam HI; M2: DNA marker (DL1000); M3: DNA marker (DL10000); M4: DNA marker (DL15000). M: Protein markers. A1~A9: uninduced <t>BL21</t> (pET32a) supernatant, uninduced BL21 (pET32a) sediment, induced BL21 (pET32a) supernatant, induced BL21 (pET32a) sediment, uninduced BL21 (P-Srr) supernatant, uninduced BL21 (P-Srr) sediment, induced BL21 (P-Srr) supernatant, induced BL21 (P-Srr) sediment, purification of recombinant rSrr. B1~B7: uninduced BL21 (pET32a), induced BL21 (pET32a), uninduced BL21 (P-NeuA), induced BL21 (P-NeuA), induced BL21 (P-NeuA) supernatant, induced BL21 (P-NeuA) sediment, purification of recombinant rNeuA. C1~C7: uninduced BL21 (pET32a), induced BL21 (pET32a), uninduced BL21 (P-Hsp), induced BL21 (P-Hsp), induced BL21 (P-Hsp) supernatant, induced BL21 (P-Hsp) sediment, purification of recombinant rHsp.
    E Coli Bl21 Cells, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Thermo Fisher bl21 ai
    Growth curve of Escherichia coli <t>BL21-AI</t> transformed with pACYCDuet-1 plasmids expressing recombinant VapB or VapC separately, or VapB and VapC together. E. coli cells carrying the empty vector were grown as control. Cells were grown in LB media at 37°C, with linear shaking (1 mm) for 15 s every 15 min. Arrow indicates expression induction by 1 mM IPTG and 0.5% (w/v) arabinose. Growth curves were initiated by adding three colonies to each well, n = 12 (each replicate consisting of three pooled colonies); error bars are standard error of mean. Results from one representative experiment, out of three with similar results are shown.
    Bl21 Ai, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/bl21 ai/product/Thermo Fisher
    Average 95 stars, based on 1 article reviews
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    KHSRP is modified by SUMO1 at the major site K87 in vitro and in cells. a-b Exogenous and endogenous KHSRP in cells are modified by SUMO1. 293T cells transfected with indicated plasmids were lysed and pulled down with Ni 2+ -NTA resin for SUMOylation assay, and SUMO1 modification of KHSRP was analyzed by Western blotting with indicated antibodies. c SUMO1 modification of KHSRP is verified by in vitro E.coli -based SUMOylation assay. Plasmid pGEX-4T-1-KHSRP was co-transformed with or without pE1E2SUMO1 plasmid into E.coli BL21 (DE3). After GST pull-down purification, Western blotting was conducted with anti-SUMO1 antibody and the same membrane was detected with anti-GST antibody after stripping. d Mutation of K87R weakens SUMO1 modification of KHSRP in 293T cells. The construct pEF-5HA-KHSRP-WT, or -K87R, or -K359R, or -K628R was co-transfected with His-SUMO1 into 293T cells. 48 h after transfection, cells were lysed for the SUMOylation assay with Ni 2+ -NTA resin

    Journal: Molecular Cancer

    Article Title: SUMO1 modification of KHSRP regulates tumorigenesis by preventing the TL-G-Rich miRNA biogenesis

    doi: 10.1186/s12943-017-0724-6

    Figure Lengend Snippet: KHSRP is modified by SUMO1 at the major site K87 in vitro and in cells. a-b Exogenous and endogenous KHSRP in cells are modified by SUMO1. 293T cells transfected with indicated plasmids were lysed and pulled down with Ni 2+ -NTA resin for SUMOylation assay, and SUMO1 modification of KHSRP was analyzed by Western blotting with indicated antibodies. c SUMO1 modification of KHSRP is verified by in vitro E.coli -based SUMOylation assay. Plasmid pGEX-4T-1-KHSRP was co-transformed with or without pE1E2SUMO1 plasmid into E.coli BL21 (DE3). After GST pull-down purification, Western blotting was conducted with anti-SUMO1 antibody and the same membrane was detected with anti-GST antibody after stripping. d Mutation of K87R weakens SUMO1 modification of KHSRP in 293T cells. The construct pEF-5HA-KHSRP-WT, or -K87R, or -K359R, or -K628R was co-transfected with His-SUMO1 into 293T cells. 48 h after transfection, cells were lysed for the SUMOylation assay with Ni 2+ -NTA resin

    Article Snippet: Briefly, pGEX-4T-1-KHSRP-WT was co-expressed with or without pE1E2SUMO1 plasmid in E.coli BL21 (DE3) respectively, and then lysed by using B-PER Protein Extraction Reagent (#78248, Thermo Fisher, USA) and incubated wi th Glutathione sepharose 4B (GE healthcare) at 4 °C overnight.

    Techniques: Modification, In Vitro, Transfection, Western Blot, Plasmid Preparation, Transformation Assay, Purification, Stripping Membranes, Mutagenesis, Construct

    Transcription-dependent loss of the target plasmid. BL21-AI strain with the STH Csm module plasmid was challenged with plasmids with STH target sequence or without a target sequence with or without IPTG (1μM) as indicated. (A) Plasmid targeting assay was performed as in Fig 2 (without arabinose). Corresponding Csm modules and targets are indicated with green boxes. (B) Presence of plasmids was assessed by plasmid extraction following overnight growth in medium with or without 1mM IPTG as indicated. Plasmid DNA was linearized, separated by agarose gel electrophoresis and visualized with ethidium bromide. Plasmid DNA from 4 individual colonies is shown for each condition. Positions of linearized Csm module and target plasmids are indicated. Dashed lines indicate rearranged non-contiguous sections of single original gel.

    Journal: PLoS ONE

    Article Title: Programmable type III-A CRISPR-Cas DNA targeting modules

    doi: 10.1371/journal.pone.0176221

    Figure Lengend Snippet: Transcription-dependent loss of the target plasmid. BL21-AI strain with the STH Csm module plasmid was challenged with plasmids with STH target sequence or without a target sequence with or without IPTG (1μM) as indicated. (A) Plasmid targeting assay was performed as in Fig 2 (without arabinose). Corresponding Csm modules and targets are indicated with green boxes. (B) Presence of plasmids was assessed by plasmid extraction following overnight growth in medium with or without 1mM IPTG as indicated. Plasmid DNA was linearized, separated by agarose gel electrophoresis and visualized with ethidium bromide. Plasmid DNA from 4 individual colonies is shown for each condition. Positions of linearized Csm module and target plasmids are indicated. Dashed lines indicate rearranged non-contiguous sections of single original gel.

    Article Snippet: Plasmid interference assay BL21-AI (Thermo Fisher Scientific) was transformed with a Csm module plasmid to generate a host strain.

    Techniques: Plasmid Preparation, Sequencing, Agarose Gel Electrophoresis

    Csm modules prevent plasmid invasion in E . coli . The BL21-AI strains containing the indicated Csm module plasmids (first column; LLA, SEP or STH) were each transformed with four target plasmids (second column; LLA, SEP, STH or vector). A series of 10-fold dilutions (10 0 to 10 6 ) of transformed cells were plated on a LB agar containing 50 μg/ml ampicillin and 34 μg/ml chloramphenicol with or without 0.2% arabinose as indicated. Representative results of 3 experiments are shown. Corresponding Csm modules and targets are indicated with green boxes. Dashed lines separate data from different plates.

    Journal: PLoS ONE

    Article Title: Programmable type III-A CRISPR-Cas DNA targeting modules

    doi: 10.1371/journal.pone.0176221

    Figure Lengend Snippet: Csm modules prevent plasmid invasion in E . coli . The BL21-AI strains containing the indicated Csm module plasmids (first column; LLA, SEP or STH) were each transformed with four target plasmids (second column; LLA, SEP, STH or vector). A series of 10-fold dilutions (10 0 to 10 6 ) of transformed cells were plated on a LB agar containing 50 μg/ml ampicillin and 34 μg/ml chloramphenicol with or without 0.2% arabinose as indicated. Representative results of 3 experiments are shown. Corresponding Csm modules and targets are indicated with green boxes. Dashed lines separate data from different plates.

    Article Snippet: Plasmid interference assay BL21-AI (Thermo Fisher Scientific) was transformed with a Csm module plasmid to generate a host strain.

    Techniques: Plasmid Preparation, Transformation Assay

    Detection of recombinant plasmids and expression and purification of recombinant proteins M1: DNA marker (DL2000); lane 1: PCR products of candidate antigen genes; lane 2: enzyme digestion of recombinant plasmids with Bam HI + Xho I; lane 3: enzyme digestion of recombinant plasmids with Bam HI; M2: DNA marker (DL1000); M3: DNA marker (DL10000); M4: DNA marker (DL15000). M: Protein markers. A1~A9: uninduced BL21 (pET32a) supernatant, uninduced BL21 (pET32a) sediment, induced BL21 (pET32a) supernatant, induced BL21 (pET32a) sediment, uninduced BL21 (P-Srr) supernatant, uninduced BL21 (P-Srr) sediment, induced BL21 (P-Srr) supernatant, induced BL21 (P-Srr) sediment, purification of recombinant rSrr. B1~B7: uninduced BL21 (pET32a), induced BL21 (pET32a), uninduced BL21 (P-NeuA), induced BL21 (P-NeuA), induced BL21 (P-NeuA) supernatant, induced BL21 (P-NeuA) sediment, purification of recombinant rNeuA. C1~C7: uninduced BL21 (pET32a), induced BL21 (pET32a), uninduced BL21 (P-Hsp), induced BL21 (P-Hsp), induced BL21 (P-Hsp) supernatant, induced BL21 (P-Hsp) sediment, purification of recombinant rHsp.

    Journal: Oncotarget

    Article Title: Identification and screening of effective protective antigens for channel catfish against Streptococcus iniae

    doi: 10.18632/oncotarget.16475

    Figure Lengend Snippet: Detection of recombinant plasmids and expression and purification of recombinant proteins M1: DNA marker (DL2000); lane 1: PCR products of candidate antigen genes; lane 2: enzyme digestion of recombinant plasmids with Bam HI + Xho I; lane 3: enzyme digestion of recombinant plasmids with Bam HI; M2: DNA marker (DL1000); M3: DNA marker (DL10000); M4: DNA marker (DL15000). M: Protein markers. A1~A9: uninduced BL21 (pET32a) supernatant, uninduced BL21 (pET32a) sediment, induced BL21 (pET32a) supernatant, induced BL21 (pET32a) sediment, uninduced BL21 (P-Srr) supernatant, uninduced BL21 (P-Srr) sediment, induced BL21 (P-Srr) supernatant, induced BL21 (P-Srr) sediment, purification of recombinant rSrr. B1~B7: uninduced BL21 (pET32a), induced BL21 (pET32a), uninduced BL21 (P-NeuA), induced BL21 (P-NeuA), induced BL21 (P-NeuA) supernatant, induced BL21 (P-NeuA) sediment, purification of recombinant rNeuA. C1~C7: uninduced BL21 (pET32a), induced BL21 (pET32a), uninduced BL21 (P-Hsp), induced BL21 (P-Hsp), induced BL21 (P-Hsp) supernatant, induced BL21 (P-Hsp) sediment, purification of recombinant rHsp.

    Article Snippet: The plasmids were then transformed into E. coli BL21 cells and induced by adding 1.0 mM IPTG at 37°C for 4 h. The cells were then centrifuged at 8000 × g for 10 min at 4°C, suspended in sterile phosphate-buffered saline (PBS), sonicated with a Sonic Dismembrator (model 500; Thermo Fisher Scientific, Waltham, MA, USA), and examined by 12.5% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).

    Techniques: Recombinant, Expressing, Purification, Marker, Polymerase Chain Reaction

    Growth curve of Escherichia coli BL21-AI transformed with pACYCDuet-1 plasmids expressing recombinant VapB or VapC separately, or VapB and VapC together. E. coli cells carrying the empty vector were grown as control. Cells were grown in LB media at 37°C, with linear shaking (1 mm) for 15 s every 15 min. Arrow indicates expression induction by 1 mM IPTG and 0.5% (w/v) arabinose. Growth curves were initiated by adding three colonies to each well, n = 12 (each replicate consisting of three pooled colonies); error bars are standard error of mean. Results from one representative experiment, out of three with similar results are shown.

    Journal: Frontiers in Microbiology

    Article Title: The vapB–vapC Operon of Acidovorax citrulli Functions as a Bona-fide Toxin–Antitoxin Module

    doi: 10.3389/fmicb.2015.01499

    Figure Lengend Snippet: Growth curve of Escherichia coli BL21-AI transformed with pACYCDuet-1 plasmids expressing recombinant VapB or VapC separately, or VapB and VapC together. E. coli cells carrying the empty vector were grown as control. Cells were grown in LB media at 37°C, with linear shaking (1 mm) for 15 s every 15 min. Arrow indicates expression induction by 1 mM IPTG and 0.5% (w/v) arabinose. Growth curves were initiated by adding three colonies to each well, n = 12 (each replicate consisting of three pooled colonies); error bars are standard error of mean. Results from one representative experiment, out of three with similar results are shown.

    Article Snippet: Escherichia coli strains DH5α , BL21(DE3) and BL21-AI (Thermo Fisher ScientificTM , Waltham, MA, USA) were routinely grown in Lysogeny broth (LB; Difco) under constant shaking (150 rpm) or LB agar (LB containing 15 g/l agar) at 37°C.

    Techniques: Transformation Assay, Expressing, Recombinant, Plasmid Preparation