Structured Review

Millipore bl21
Analysis of fusion protein by SDS-PAGE and Western blot. (a) Expression and purification of IFN-CSP. Lane M: protein molecular weight marker. Lanes 1 and 2: total proteins of E. coli <t>BL21/pET-21b-IFN-CSP</t> before and after induction. Lanes 3 and 4: supernatant and precipitation after ultrasonication and centrifugation. Lane 5: purified IFN-CSP using Ni affinity chromatography. Lane 6: Purified IFN-CSP using HiTrap affinity chromatography. (b) IFN-CSP was analyzed by SDS-PAGE, transferred to PVDF membrane, and detected by goat polyclonal anti-human IFN α antibody. Lane M: protein molecular weight marker. Lanes 1 and 2: total proteins of E. coli BL21/pET-21b-IFN-CSP before and after induction.
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Images

1) Product Images from "Construction of a Novel Liver-Targeting Fusion Interferon by Incorporation of a Plasmodium Region I-Plus Peptide"

Article Title: Construction of a Novel Liver-Targeting Fusion Interferon by Incorporation of a Plasmodium Region I-Plus Peptide

Journal: BioMed Research International

doi: 10.1155/2014/261631

Analysis of fusion protein by SDS-PAGE and Western blot. (a) Expression and purification of IFN-CSP. Lane M: protein molecular weight marker. Lanes 1 and 2: total proteins of E. coli BL21/pET-21b-IFN-CSP before and after induction. Lanes 3 and 4: supernatant and precipitation after ultrasonication and centrifugation. Lane 5: purified IFN-CSP using Ni affinity chromatography. Lane 6: Purified IFN-CSP using HiTrap affinity chromatography. (b) IFN-CSP was analyzed by SDS-PAGE, transferred to PVDF membrane, and detected by goat polyclonal anti-human IFN α antibody. Lane M: protein molecular weight marker. Lanes 1 and 2: total proteins of E. coli BL21/pET-21b-IFN-CSP before and after induction.
Figure Legend Snippet: Analysis of fusion protein by SDS-PAGE and Western blot. (a) Expression and purification of IFN-CSP. Lane M: protein molecular weight marker. Lanes 1 and 2: total proteins of E. coli BL21/pET-21b-IFN-CSP before and after induction. Lanes 3 and 4: supernatant and precipitation after ultrasonication and centrifugation. Lane 5: purified IFN-CSP using Ni affinity chromatography. Lane 6: Purified IFN-CSP using HiTrap affinity chromatography. (b) IFN-CSP was analyzed by SDS-PAGE, transferred to PVDF membrane, and detected by goat polyclonal anti-human IFN α antibody. Lane M: protein molecular weight marker. Lanes 1 and 2: total proteins of E. coli BL21/pET-21b-IFN-CSP before and after induction.

Techniques Used: SDS Page, Western Blot, Expressing, Purification, Molecular Weight, Marker, Positron Emission Tomography, Centrifugation, Affinity Chromatography

2) Product Images from "Host cell and expression engineering for development of an E. coli ketoreductase catalyst: Enhancement of formate dehydrogenase activity for regeneration of NADH"

Article Title: Host cell and expression engineering for development of an E. coli ketoreductase catalyst: Enhancement of formate dehydrogenase activity for regeneration of NADH

Journal: Microbial Cell Factories

doi: 10.1186/1475-2859-11-7

Bioreductions of D-xylose catalyzed by whole cells and crude cell extracts of E. coli strains BL21_XR_FDH and Rosetta_XR_2FDH . Type of catalyst is indicated by symbols: ● Rosetta_XR_2FDH crude extract; ○ Rosetta_XR_2FDH whole-cell catalyst; ∇ BL21_XR_FDH crude extract; ▼ BL21_XR_FDH whole-cell catalyst. Reaction conditions: D-xylose (250 mM), sodium formate (300 mM), cells (10 g CDW /L), 30°C.
Figure Legend Snippet: Bioreductions of D-xylose catalyzed by whole cells and crude cell extracts of E. coli strains BL21_XR_FDH and Rosetta_XR_2FDH . Type of catalyst is indicated by symbols: ● Rosetta_XR_2FDH crude extract; ○ Rosetta_XR_2FDH whole-cell catalyst; ∇ BL21_XR_FDH crude extract; ▼ BL21_XR_FDH whole-cell catalyst. Reaction conditions: D-xylose (250 mM), sodium formate (300 mM), cells (10 g CDW /L), 30°C.

Techniques Used:

3) Product Images from "Soluble Expression of Humanized Anti-CD20 Single Chain Antibody in Escherichia coli by Cytoplasmic Chaperones Co-expression"

Article Title: Soluble Expression of Humanized Anti-CD20 Single Chain Antibody in Escherichia coli by Cytoplasmic Chaperones Co-expression

Journal: Avicenna Journal of Medical Biotechnology

doi:

SDS-PAGE analysis of the purified recombinant huscFv in BL21 (DE3) after gel filtration chromatography and washing with buffer A (5 mM and 20 mM of imidazole). Lane 1: the standard protein weight marker Lane 2: purified huscFv/pTf16 Lane 3: purified huscFv/pGro7 Lane 4: purified huscFv/pG-KJE8 Lane 5: purified huscFv/pKjE7 Lane 6: purified huscFv/pG-Tf2
Figure Legend Snippet: SDS-PAGE analysis of the purified recombinant huscFv in BL21 (DE3) after gel filtration chromatography and washing with buffer A (5 mM and 20 mM of imidazole). Lane 1: the standard protein weight marker Lane 2: purified huscFv/pTf16 Lane 3: purified huscFv/pGro7 Lane 4: purified huscFv/pG-KJE8 Lane 5: purified huscFv/pKjE7 Lane 6: purified huscFv/pG-Tf2

Techniques Used: SDS Page, Purification, Recombinant, Filtration, Chromatography, Marker

4) Product Images from "Soluble Expression of Humanized Anti-CD20 Single Chain Antibody in Escherichia coli by Cytoplasmic Chaperones Co-expression"

Article Title: Soluble Expression of Humanized Anti-CD20 Single Chain Antibody in Escherichia coli by Cytoplasmic Chaperones Co-expression

Journal: Avicenna Journal of Medical Biotechnology

doi:

SDS–PAGE analysis of co-expressed anti-CD20 huscFv with different cytoplasmic molecular chaperones after induction with 0.4 Mm IPTG for 4 hr at 30° C , washed and resuspended in lysis buffer 10 ml of lysis buffer (100 mM NaCl, 50 mM NaH2PO4 at pH=8.0). Lane 1: the standard protein weight marker. Lane 2: whole cell lysate supernatant from uninduced cells without chaperone plasmid set. Lane 3: whole insoluble pellet from uninduced cells without chaperone plasmid set. Lanes 4–6: samples of different extracts from E. coli BL21 (DE3)/pET22b-huscFv/pG-KJE8 (Lane 4: supernatant from uninduced cells, Lane 5: supernatant from 4 hr , Lane 6: pellet from 4 hr ). Lanes7–9: samples of different extracts from E. coli BL21 (DE3)/pET22b-huscFv/pGro7 (Lane 7: supernatant from uninduced cells, Lane 8: supernatant from 4 hr , Lane 9: pellet from 4 hr ). Lanes 10–12: samples of different extracts from E. coli BL21 (DE3)/pET22b-huscFv/pKjE7 (Lane 10: supernatant from uninduced cells, Lane 11: supernatant from 4 hr , Lane 12: pellet from 4 hr ). Lanes 13–15: samples of different extracts from E. coli BL21 (DE3)/pET22b-huscFv/pG-Tf2 (Lane 13: supernatant from uninduced cells, Lane 14: supernatant from 4 hr , Lane 15: pellet from 4 hr ). Lanes 16–18: samples of different extracts from E. coli BL21 (DE3)/pET22b-husc Fv/pTf16 (Lane 16: supernatant from uninduced cells, Lane 17: supernatant from 4 hr , Lane 18: pellet from 4 hr ).
Figure Legend Snippet: SDS–PAGE analysis of co-expressed anti-CD20 huscFv with different cytoplasmic molecular chaperones after induction with 0.4 Mm IPTG for 4 hr at 30° C , washed and resuspended in lysis buffer 10 ml of lysis buffer (100 mM NaCl, 50 mM NaH2PO4 at pH=8.0). Lane 1: the standard protein weight marker. Lane 2: whole cell lysate supernatant from uninduced cells without chaperone plasmid set. Lane 3: whole insoluble pellet from uninduced cells without chaperone plasmid set. Lanes 4–6: samples of different extracts from E. coli BL21 (DE3)/pET22b-huscFv/pG-KJE8 (Lane 4: supernatant from uninduced cells, Lane 5: supernatant from 4 hr , Lane 6: pellet from 4 hr ). Lanes7–9: samples of different extracts from E. coli BL21 (DE3)/pET22b-huscFv/pGro7 (Lane 7: supernatant from uninduced cells, Lane 8: supernatant from 4 hr , Lane 9: pellet from 4 hr ). Lanes 10–12: samples of different extracts from E. coli BL21 (DE3)/pET22b-huscFv/pKjE7 (Lane 10: supernatant from uninduced cells, Lane 11: supernatant from 4 hr , Lane 12: pellet from 4 hr ). Lanes 13–15: samples of different extracts from E. coli BL21 (DE3)/pET22b-huscFv/pG-Tf2 (Lane 13: supernatant from uninduced cells, Lane 14: supernatant from 4 hr , Lane 15: pellet from 4 hr ). Lanes 16–18: samples of different extracts from E. coli BL21 (DE3)/pET22b-husc Fv/pTf16 (Lane 16: supernatant from uninduced cells, Lane 17: supernatant from 4 hr , Lane 18: pellet from 4 hr ).

Techniques Used: SDS Page, Lysis, Marker, Plasmid Preparation

SDS-PAGE analysis of the purified recombinant huscFv in BL21 (DE3) after gel filtration chromatography and washing with buffer A (5 mM and 20 mM of imidazole). Lane 1: the standard protein weight marker Lane 2: purified huscFv/pTf16 Lane 3: purified huscFv/pGro7 Lane 4: purified huscFv/pG-KJE8 Lane 5: purified huscFv/pKjE7 Lane 6: purified huscFv/pG-Tf2
Figure Legend Snippet: SDS-PAGE analysis of the purified recombinant huscFv in BL21 (DE3) after gel filtration chromatography and washing with buffer A (5 mM and 20 mM of imidazole). Lane 1: the standard protein weight marker Lane 2: purified huscFv/pTf16 Lane 3: purified huscFv/pGro7 Lane 4: purified huscFv/pG-KJE8 Lane 5: purified huscFv/pKjE7 Lane 6: purified huscFv/pG-Tf2

Techniques Used: SDS Page, Purification, Recombinant, Filtration, Chromatography, Marker

5) Product Images from "A Molecular Biological and Biochemical Investigation on Mycobacterium tuberculosis MutT Protein"

Article Title: A Molecular Biological and Biochemical Investigation on Mycobacterium tuberculosis MutT Protein

Journal: Jundishapur Journal of Microbiology

doi: 10.5812/jjm.9367

Characterization of the Anti- M. tuberculosis MutT Antisera A. Titers of antisera prepared from the same rabbit immunized with purified M. tuberculosis MutT are examined using total lysate of E. coli strain BL21 (DE3) harboring pET28a (+)-mutT. The batches of antisera were collected on the 6th (1st) and the 7th day (2nd) after the 1st and the 2nd boost immunization, respectively. A prominent signal of the recombinant M. tuberculosis MutT protein was still present (arrowhead) even at high dilution factors. B. The same blots were stripped and probed with anti-His antibody (Clontech, 1:5000) to mark the position of the recombinant M. tuberculosis MutT protein.
Figure Legend Snippet: Characterization of the Anti- M. tuberculosis MutT Antisera A. Titers of antisera prepared from the same rabbit immunized with purified M. tuberculosis MutT are examined using total lysate of E. coli strain BL21 (DE3) harboring pET28a (+)-mutT. The batches of antisera were collected on the 6th (1st) and the 7th day (2nd) after the 1st and the 2nd boost immunization, respectively. A prominent signal of the recombinant M. tuberculosis MutT protein was still present (arrowhead) even at high dilution factors. B. The same blots were stripped and probed with anti-His antibody (Clontech, 1:5000) to mark the position of the recombinant M. tuberculosis MutT protein.

Techniques Used: Purification, Recombinant

Overexpression of the Recombinant M. tuberculosis MutT Protein in BL21 (DE3) Plasmid pET28a (+)-mutT was overexpressed in E. coli strain BL21 (DE3) upon IPTG induction to produce the recombinant M. tuberculosis MutT protein. Lysates of bacteria were fractionated in a SDS-PAGE gel, and stained with Coomassie Blue. The recombinant M. tuberculosis MutT protein of 17.3 kD was found in the fractions.
Figure Legend Snippet: Overexpression of the Recombinant M. tuberculosis MutT Protein in BL21 (DE3) Plasmid pET28a (+)-mutT was overexpressed in E. coli strain BL21 (DE3) upon IPTG induction to produce the recombinant M. tuberculosis MutT protein. Lysates of bacteria were fractionated in a SDS-PAGE gel, and stained with Coomassie Blue. The recombinant M. tuberculosis MutT protein of 17.3 kD was found in the fractions.

Techniques Used: Over Expression, Recombinant, Plasmid Preparation, SDS Page, Staining

6) Product Images from "Preparation of Biologically Active Single-Chain Variable Antibody Fragments that Target the HIV-1 gp120 V3 Loop"

Article Title: Preparation of Biologically Active Single-Chain Variable Antibody Fragments that Target the HIV-1 gp120 V3 Loop

Journal: Cellular and molecular biology (Noisy-le-Grand, France)

doi:

Schematic representation of the purification and refolding of KD-247 scFv. KD-247 scFv was overexpressed in the BL21 (DE3) strain using the pET system. The pellet recovered after cell lysis was washed twice before denaturation with 6 M Gu-HCl containing
Figure Legend Snippet: Schematic representation of the purification and refolding of KD-247 scFv. KD-247 scFv was overexpressed in the BL21 (DE3) strain using the pET system. The pellet recovered after cell lysis was washed twice before denaturation with 6 M Gu-HCl containing

Techniques Used: Purification, Positron Emission Tomography, Lysis

7) Product Images from "Production of bioactive liver-targeting interferon Mu-IFN-CSP by soluble prokaryotic expression"

Article Title: Production of bioactive liver-targeting interferon Mu-IFN-CSP by soluble prokaryotic expression

Journal: AMB Express

doi: 10.1186/s13568-017-0493-z

Schematic diagram of mu-IFN-CSP gene in the expression vector mu-IFN-CSP/pET-21b and expression of mu-IFN-CSP protein in recombinant E. coli . a A schematic diagram of mu-IFN-CSP/pET-21b. b SDS-PAGE analysis of mu-IFN-CSP expression. Lane 1 total proteins of recombinant E. coli BL21 after IPTG induction. Lanes 2–3 supernatant and precipitation after ultrasonication and centrifugation. Lane M protein molecular weight marker. c Recombinant mu-IFN-CSP was analyzed by western blot analysis. Lanes 1–2 total proteins of E. coli BL21/pET-21b-mu-IFN-CSP before and after induction. Lane M protein molecular weight marker
Figure Legend Snippet: Schematic diagram of mu-IFN-CSP gene in the expression vector mu-IFN-CSP/pET-21b and expression of mu-IFN-CSP protein in recombinant E. coli . a A schematic diagram of mu-IFN-CSP/pET-21b. b SDS-PAGE analysis of mu-IFN-CSP expression. Lane 1 total proteins of recombinant E. coli BL21 after IPTG induction. Lanes 2–3 supernatant and precipitation after ultrasonication and centrifugation. Lane M protein molecular weight marker. c Recombinant mu-IFN-CSP was analyzed by western blot analysis. Lanes 1–2 total proteins of E. coli BL21/pET-21b-mu-IFN-CSP before and after induction. Lane M protein molecular weight marker

Techniques Used: Expressing, Plasmid Preparation, Positron Emission Tomography, Recombinant, SDS Page, Centrifugation, Molecular Weight, Marker, Western Blot

8) Product Images from "Zinc Metalloproteinase ProA Directly Activates Legionella pneumophila PlaC Glycerophospholipid:cholesterol Acyltransferase *"

Article Title: Zinc Metalloproteinase ProA Directly Activates Legionella pneumophila PlaC Glycerophospholipid:cholesterol Acyltransferase *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M112.346387

L. pneumophila zinc metalloproteinase ProA directly activates PlaC GCAT activity transferring both palmitic and propionic acid to cholesterol and analysis of acyl donor substrate specificities. A , E. coli BL21 cell lysates from clones expressing plaC
Figure Legend Snippet: L. pneumophila zinc metalloproteinase ProA directly activates PlaC GCAT activity transferring both palmitic and propionic acid to cholesterol and analysis of acyl donor substrate specificities. A , E. coli BL21 cell lysates from clones expressing plaC

Techniques Used: Activity Assay, Transferring, Clone Assay, Expressing

9) Product Images from "Bacterial Genome Partitioning: N-Terminal Domain of IncC Protein Encoded by Broad-Host-Range Plasmid RK2 Modulates Oligomerisation and DNA Binding"

Article Title: Bacterial Genome Partitioning: N-Terminal Domain of IncC Protein Encoded by Broad-Host-Range Plasmid RK2 Modulates Oligomerisation and DNA Binding

Journal: Journal of Molecular Biology

doi: 10.1016/j.jmb.2008.12.016

Purification and oligomerisation of IncC1/IncC2. (a) Overexpression and purification of IncC: (i) Comparison of the overexpression of His-IncC1 (C1) and IncC2 (C2) in BL21 and with decreasing IPTG concentrations of 1, 0.5, 0.1 and 0 mM. (ii) Elution samples from nickel-agarose column. (b) Glutaraldehyde cross-linking of IncC1 and IncC2 in the presence of nucleotides. Triangles indicate the increasing percentage of glutaraldehyde added (0, 0.001%, 0.01%, 0.05% and 0.1%; except for ATPγS and NTD, which have no 0.001%). Samples were separated by 9% SDS-PAGE, except for the NTD sample, which was separated by 14% SDS-PAGE. Molecular weight markers are in kilodaltons. HO indicates higher-order structures; C1, IncC1; C2, IncC2; and N, NTD. Subscripts denote the oligomeric state of the proteins. (c) Pelleting studies to show the solubility of IncC1 and IncC2 in the absence (0) or presence of 2 mM nucleotides. S indicates soluble; I, insoluble.
Figure Legend Snippet: Purification and oligomerisation of IncC1/IncC2. (a) Overexpression and purification of IncC: (i) Comparison of the overexpression of His-IncC1 (C1) and IncC2 (C2) in BL21 and with decreasing IPTG concentrations of 1, 0.5, 0.1 and 0 mM. (ii) Elution samples from nickel-agarose column. (b) Glutaraldehyde cross-linking of IncC1 and IncC2 in the presence of nucleotides. Triangles indicate the increasing percentage of glutaraldehyde added (0, 0.001%, 0.01%, 0.05% and 0.1%; except for ATPγS and NTD, which have no 0.001%). Samples were separated by 9% SDS-PAGE, except for the NTD sample, which was separated by 14% SDS-PAGE. Molecular weight markers are in kilodaltons. HO indicates higher-order structures; C1, IncC1; C2, IncC2; and N, NTD. Subscripts denote the oligomeric state of the proteins. (c) Pelleting studies to show the solubility of IncC1 and IncC2 in the absence (0) or presence of 2 mM nucleotides. S indicates soluble; I, insoluble.

Techniques Used: Purification, Over Expression, SDS Page, Molecular Weight, Solubility

10) Product Images from "The pCri System: A Vector Collection for Recombinant Protein Expression and Purification"

Article Title: The pCri System: A Vector Collection for Recombinant Protein Expression and Purification

Journal: PLoS ONE

doi: 10.1371/journal.pone.0112643

Protein expression and purification trials using the pCri System. ( A ) The GFP gene was cloned into pCri-1a, 4a, 6a, 8a, 11a, and 14a, the proteins expressed in E. coli BL21 cells, and subsequently purified by Ni-NTA-affinity chromatography except for MBP, GST, and LSL fusion products, which were purified by their respective specific affinity resins. ( B ) The gene coding for fragilysin was cloned into pCri-1a, 4a, 6a and 8a, and expressed in E. coli Origami 2 cells. Total (T) and soluble (S) fractions of crude protein extracts were further analysed by SDS-PAGE. All expression trials were performed at 20°C except for pCri-1a, which was also performed at 37°C. ( C ) Partially purified MBP-fragilysin before (−) and after (+) TEV proteinase cleavage. Arrows indicate the soluble fraction of fragilysin (white) and the MBP (black) after TEV proteinase cleavage. ( D ) Expression of CPA2 intracellularly (lanes 1 and 2) or periplasmatically (lanes 3 and 4) in E. coli cells, and extracellularly (lanes 5 and 6) in P. pastoris cells. Lanes indicate samples before (1, 3 and 5) and after (2, 4 and 6) tryptic digestion. Arrows indicate the pro-CPA2 (black), the mature form (grey) and the pro-peptide (white) after tryptic cleavage. ( E ) The PNGase F gene was cloned into pCri-4a and 8a and expressed overnight at 20°C in E. coli BL21 and Origami 2 cells. Total (T) and soluble (S) fractions of crude protein extracts were further analysed by SDS-PAGE. ( F ) Activity of affinity-purified TRX-PNGase F against glycosylated RNase B. (+) and (−) indicate presence and absence of PNGase F. Arrows indicate the PNGase F (black), native RNase B (grey) and deglycosylated RNase B (white). ( G ) MecR1 was expressed in E. coli BL21 using pCri-8a or 13a, and soluble fractions were analysed by Western blotting with specific antibodies as detailed in “ Materials and Methods ”. A black arrow indicates the detected MecR1. ( H ) Partially purified MISTIC-MecR1 after Ni-NTA-affinity purification. ( I ) Partially purified MBP-GFP, SUMO-GFP and MISTIC-MecR1 were digested with TEV proteinase, SENP1 or thrombin, respectively. For TEV proteinase and SENP1 digestions various ratios of proteinase∶tagged-protein were tested in overnight incubations at 4°C, whereas for thrombin digestions 2 units of proteinase were used to digest 25 µg of protein for various times at room temperature. Arrows indicate tagged-protein (black), target protein (grey) and fused-tag (white) after proteinase cleavage.
Figure Legend Snippet: Protein expression and purification trials using the pCri System. ( A ) The GFP gene was cloned into pCri-1a, 4a, 6a, 8a, 11a, and 14a, the proteins expressed in E. coli BL21 cells, and subsequently purified by Ni-NTA-affinity chromatography except for MBP, GST, and LSL fusion products, which were purified by their respective specific affinity resins. ( B ) The gene coding for fragilysin was cloned into pCri-1a, 4a, 6a and 8a, and expressed in E. coli Origami 2 cells. Total (T) and soluble (S) fractions of crude protein extracts were further analysed by SDS-PAGE. All expression trials were performed at 20°C except for pCri-1a, which was also performed at 37°C. ( C ) Partially purified MBP-fragilysin before (−) and after (+) TEV proteinase cleavage. Arrows indicate the soluble fraction of fragilysin (white) and the MBP (black) after TEV proteinase cleavage. ( D ) Expression of CPA2 intracellularly (lanes 1 and 2) or periplasmatically (lanes 3 and 4) in E. coli cells, and extracellularly (lanes 5 and 6) in P. pastoris cells. Lanes indicate samples before (1, 3 and 5) and after (2, 4 and 6) tryptic digestion. Arrows indicate the pro-CPA2 (black), the mature form (grey) and the pro-peptide (white) after tryptic cleavage. ( E ) The PNGase F gene was cloned into pCri-4a and 8a and expressed overnight at 20°C in E. coli BL21 and Origami 2 cells. Total (T) and soluble (S) fractions of crude protein extracts were further analysed by SDS-PAGE. ( F ) Activity of affinity-purified TRX-PNGase F against glycosylated RNase B. (+) and (−) indicate presence and absence of PNGase F. Arrows indicate the PNGase F (black), native RNase B (grey) and deglycosylated RNase B (white). ( G ) MecR1 was expressed in E. coli BL21 using pCri-8a or 13a, and soluble fractions were analysed by Western blotting with specific antibodies as detailed in “ Materials and Methods ”. A black arrow indicates the detected MecR1. ( H ) Partially purified MISTIC-MecR1 after Ni-NTA-affinity purification. ( I ) Partially purified MBP-GFP, SUMO-GFP and MISTIC-MecR1 were digested with TEV proteinase, SENP1 or thrombin, respectively. For TEV proteinase and SENP1 digestions various ratios of proteinase∶tagged-protein were tested in overnight incubations at 4°C, whereas for thrombin digestions 2 units of proteinase were used to digest 25 µg of protein for various times at room temperature. Arrows indicate tagged-protein (black), target protein (grey) and fused-tag (white) after proteinase cleavage.

Techniques Used: Expressing, Purification, Clone Assay, Affinity Chromatography, SDS Page, Activity Assay, Affinity Purification, Western Blot

11) Product Images from "Immunization with Recombinant Transferrin Binding Protein B Enhances Clearance of Nontypeable Haemophilus influenzae from the Rat Lung"

Article Title: Immunization with Recombinant Transferrin Binding Protein B Enhances Clearance of Nontypeable Haemophilus influenzae from the Rat Lung

Journal: Infection and Immunity

doi:

SDS-PAGE of samples from the purification of rTbpB and the corresponding transferrin dot enzyme assay. The GST-rTbpB fusion protein is present in the cell lysate of the IPTG-induced culture of BL21/pCU17 (arrow) in lane 2 compared to the uninduced lysate in lane 1. The fusion protein binds to glutathione-Sepharose (lane 3), although some free GST (∼26 kDa), probably resulting from endogenous Escherichia coli protease activity, is also bound. rTbpB (starred arrow) is released from the Sepharose after thrombin cleavage (lane 4) and further purified by cation-exchange high-pressure liquid chromatography (lane 5). The lower panel demonstrates that samples in lanes 2 to 5 have transferrin binding activity, although the activity in the induced E. coli cell lysate (lane 2) was weak.
Figure Legend Snippet: SDS-PAGE of samples from the purification of rTbpB and the corresponding transferrin dot enzyme assay. The GST-rTbpB fusion protein is present in the cell lysate of the IPTG-induced culture of BL21/pCU17 (arrow) in lane 2 compared to the uninduced lysate in lane 1. The fusion protein binds to glutathione-Sepharose (lane 3), although some free GST (∼26 kDa), probably resulting from endogenous Escherichia coli protease activity, is also bound. rTbpB (starred arrow) is released from the Sepharose after thrombin cleavage (lane 4) and further purified by cation-exchange high-pressure liquid chromatography (lane 5). The lower panel demonstrates that samples in lanes 2 to 5 have transferrin binding activity, although the activity in the induced E. coli cell lysate (lane 2) was weak.

Techniques Used: SDS Page, Purification, Enzymatic Assay, Activity Assay, High Performance Liquid Chromatography, Binding Assay

12) Product Images from "YeeV is an Escherichia coli Toxin that Inhibits Cell Division by Targeting the Cytoskeleton Proteins, FtsZ and MreB"

Article Title: YeeV is an Escherichia coli Toxin that Inhibits Cell Division by Targeting the Cytoskeleton Proteins, FtsZ and MreB

Journal: Molecular microbiology

doi: 10.1111/j.1365-2958.2010.07433.x

Ectopic expression of yeeV affects cell growth and cell shape (A). Growth curve shows cell growth inhibition after over-expression of YeeV. E. coli BW25113 cells harboring plasmid pBAD33- yeeV were grown at 37° C in glycerol M9 medium in the presence of 0.2% arabinose. (B).Phase contrast images of E.coli BW25113 cells carrying pBAD33-yeeV shows altered cell morphology after over-expression of YeeV. Cells were grown and induced as in (A). (C). Morphological manifestation of inhibition of FtsZ and MreB 1. E. coli BL21 (DE3) carrying both pET28c- sulA and pBAD33- yeeV were grown as described in (A). 1. MreB was inhibited by A22 (5μg/ml). 2. FtsZ was inhibited by SulA induced from pET28c by adding 1mM IPTG. 3. After one hour induction of SulA, cells were treated with A22 (5 μg/ml) to inhibit MreB. 4. After one hour induction of SulA, YeeV was induced from pBAD33 by adding 0.2% arabinose. All the bars represent 2 μm.
Figure Legend Snippet: Ectopic expression of yeeV affects cell growth and cell shape (A). Growth curve shows cell growth inhibition after over-expression of YeeV. E. coli BW25113 cells harboring plasmid pBAD33- yeeV were grown at 37° C in glycerol M9 medium in the presence of 0.2% arabinose. (B).Phase contrast images of E.coli BW25113 cells carrying pBAD33-yeeV shows altered cell morphology after over-expression of YeeV. Cells were grown and induced as in (A). (C). Morphological manifestation of inhibition of FtsZ and MreB 1. E. coli BL21 (DE3) carrying both pET28c- sulA and pBAD33- yeeV were grown as described in (A). 1. MreB was inhibited by A22 (5μg/ml). 2. FtsZ was inhibited by SulA induced from pET28c by adding 1mM IPTG. 3. After one hour induction of SulA, cells were treated with A22 (5 μg/ml) to inhibit MreB. 4. After one hour induction of SulA, YeeV was induced from pBAD33 by adding 0.2% arabinose. All the bars represent 2 μm.

Techniques Used: Expressing, Inhibition, Over Expression, Plasmid Preparation

Related Articles

Clone Assay:

Article Title: A simple vector system to improve performance and utilisation of recombinant antibodies
Article Snippet: Paragraph title: Reagents, scFv clones & bacterial strains ... E. coli BL21 (DE3) and Origami 2™ (DE3) cells and the pET26b(+) plasmid were from Novagen (San Diego, CA).

Article Title: Genomic and molecular characterization of a novel quorum sensing molecule in Bacillus licheniformis
Article Snippet: The expression strain [E. coli BL21 (DE3)] and E. coli TOP10 were used for cloning/transformations and were selected on LBA supplemented with ampicilin (100 µgml−1 ). .. E. coli BL21 ComX producer strain was cultivated in M9 minimal salts solution (sigma).

Article Title: The gaf Fimbrial Gene Cluster of Escherichia coli Expresses a Full-Size and a Truncated Soluble Adhesin Protein
Article Snippet: Plasmid pRR-5 contains the complete gaf gene cluster from E. coli strain IHE11165 on a 7-kb DNA fragment in pACYC184 , and pHUB110 contains a 6-bp in-frame deletion within the coding region of gafD , resulting in G fimbriae lacking GlcNAc-binding capacity ( ). pHUB113 ( ) contains the gafD reading frame cloned into pUC19. .. E. coli strain BL21 λDE3 ( ompT lon ) and expression vector pET-22b(+) were from Novagen Inc. (Madison, Wis.).

Centrifugation:

Article Title: A new hybrid bacteriocin, Ent35-MccV, displays antimicrobial activity against pathogenic Gram-positive and Gram-negative bacteria
Article Snippet: 2.3 Gene expression and bacteriocins purification for activity determinations Ent35–MccV was expressed from pMA24 in E. coli BL21 [DE3] (pLysS) grown in LB (Sigma) at 37 °C. .. After 3 h, cells were collected by centrifugation in 10 mM HEPES, pH 7, PMSF 0.5 mM (buffer A), lysed using a French press and centrifuged at 47,000xg , 4 °C for 1 h. The supernatant was heated (10 min, 100 °C) and centrifuged (10 min, 10,000xg ).

Article Title: Hypoallergenic Der p 1/Der p 2 combination vaccines for immunotherapy of house dust mite allergy
Article Snippet: Expression plasmids containing the Der p 2/1 constructs were transformed into E coli strain BL21 (DE3; Novagen, Merck). .. Protein expression was performed in 250 mL of liquid culture by means of induction with 0.5 mmol/L isopropyl-β-D-1-thiogalactopyranoside at an OD600 of 0.8 for 4 hours at 37°C, and cells were harvested by means of centrifugation at 4000 g for 15 minutes at 4°C.

Article Title: Purification and Reconstitution of the Connexin43 Carboxyl Terminus attached to the 4th Transmembrane Domain in Detergent Micelles *
Article Snippet: The E. coli strains BL21(DE3) (Novagen), Rosetta-2(DE3) (Novagen), C41(DE3) (Lucigen, ( )), and C43(DE3) (Lucigen, ( )) were transformed with the TM4-Cx43CT expression plasmid (see above) and then inoculated into 1 L of Luria Bertani medium (LB), enriched minimal media , or ISOGRO media (Sigma-Aldrich). .. The cells were harvested by centrifugation (1,000 × g for 45 min), washed with PBS buffer, and stored at −20°C.

Positron Emission Tomography:

Article Title: A Single Codon Optimization Enhances Recombinant Human TNF-α Vaccine Expression in Escherichia coli
Article Snippet: .. The vector pET-22b and E. coli strain BL21 (DE3) were purchased from Novagen (San Diego, CA). .. Isopropyl β -D-1-thiogalactopyranoside (IPTG) was from Sigma-Aldrich (St. Louis, MO).

Article Title: The gaf Fimbrial Gene Cluster of Escherichia coli Expresses a Full-Size and a Truncated Soluble Adhesin Protein
Article Snippet: .. E. coli strain BL21 λDE3 ( ompT lon ) and expression vector pET-22b(+) were from Novagen Inc. (Madison, Wis.). .. The bacteria were cultivated at 37°C in Luria broth ( ) containing the appropriate antibiotics.

Article Title: Identification and molecular characterization of a metagenome-derived L-lysine decarboxylase gene from subtropical soil microorganisms
Article Snippet: .. Plasmid pET-30a(+) (Novagen) and bacterial strain E . coli BL21 (DE3) pLysS (Novagen) were used as the expression vector and host, respectively. ..

Ligation:

Article Title: Chemical complexity of protein determines optimal E. coli expression host; A comparative study using Erythropoietin, Streptokinase and Tumor Necrosis Factor Receptor
Article Snippet: 2.1 Bacterial strains and plasmids For initial transformation of the ligation mixtures, the maintenance strain E. coli DH5α was used. .. E. coli BL21 (DE3), E. coli BL21 (DE3) pLys S, E. coli BL21 Rosetta (DE3) pLys S (Novagen,) are IPTG inducible strains in which the T7 RNA Polymerase gene is under the control of a Lac UV5 promoter.

Mouse Assay:

Article Title: A novel HPV16 E7-affitoxin for targeted therapy of HPV16-induced human cervical cancer
Article Snippet: ICR mice, weighing 23-27 g, were purchased from the animal experimental center of Wenzhou Medical University, China. .. The pET21a(+) vector and E.coli BL21 (DE3) were purchased from Novagen and ATCC, respectively.

Synthesized:

Article Title: Fluorescence Resonance Energy Transfer Assay for High-Throughput Screening of ADAMTS1 Inhibitors
Article Snippet: Materials E . coli BL21 (DE3), E. coli DH5a, pET32a(+) vector and his-bind purification kit were purchased from Novagen. .. The peptide, Abz-YPLPRNITEGEARGNVILTAK(Dnp)P-OH, was synthesized by GL Biochem Ltd. (Shanghai, China).

Article Title: Preparation and Antitumor Activity of CS5931, A Novel Polypeptide from Sea Squirt Ciona Savignyi
Article Snippet: .. Recombinant Expression of CS5931 from E. coli BL21 (DE3) The expression vector, pET28a/CS5931 synthesized by our lab previously [ ], was transfected into E. coli BL21 (DE3) cells (Novagen, Darmstadt, Germany). .. Cells were incubated in low-salt LB medium (containing 100 µg/mL Kanamycin) at 37 °C with shaking at 200 rpm.

Transfection:

Article Title: Preparation and Antitumor Activity of CS5931, A Novel Polypeptide from Sea Squirt Ciona Savignyi
Article Snippet: .. Recombinant Expression of CS5931 from E. coli BL21 (DE3) The expression vector, pET28a/CS5931 synthesized by our lab previously [ ], was transfected into E. coli BL21 (DE3) cells (Novagen, Darmstadt, Germany). .. Cells were incubated in low-salt LB medium (containing 100 µg/mL Kanamycin) at 37 °C with shaking at 200 rpm.

Construct:

Article Title: Hypoallergenic Der p 1/Der p 2 combination vaccines for immunotherapy of house dust mite allergy
Article Snippet: .. Expression plasmids containing the Der p 2/1 constructs were transformed into E coli strain BL21 (DE3; Novagen, Merck). .. Protein expression was performed in 250 mL of liquid culture by means of induction with 0.5 mmol/L isopropyl-β-D-1-thiogalactopyranoside at an OD600 of 0.8 for 4 hours at 37°C, and cells were harvested by means of centrifugation at 4000 g for 15 minutes at 4°C.

Purification:

Article Title: A new hybrid bacteriocin, Ent35-MccV, displays antimicrobial activity against pathogenic Gram-positive and Gram-negative bacteria
Article Snippet: .. 2.3 Gene expression and bacteriocins purification for activity determinations Ent35–MccV was expressed from pMA24 in E. coli BL21 [DE3] (pLysS) grown in LB (Sigma) at 37 °C. ..

Article Title: Hypoallergenic Der p 1/Der p 2 combination vaccines for immunotherapy of house dust mite allergy
Article Snippet: Paragraph title: Expression and purification of Der p 2/1 mosaic proteins ... Expression plasmids containing the Der p 2/1 constructs were transformed into E coli strain BL21 (DE3; Novagen, Merck).

Article Title: Fluorescence Resonance Energy Transfer Assay for High-Throughput Screening of ADAMTS1 Inhibitors
Article Snippet: .. Materials E . coli BL21 (DE3), E. coli DH5a, pET32a(+) vector and his-bind purification kit were purchased from Novagen. .. Heparin sepharose CL-6B column was purchased from Amersham Biosciences.

Article Title: Purification and Reconstitution of the Connexin43 Carboxyl Terminus attached to the 4th Transmembrane Domain in Detergent Micelles *
Article Snippet: Paragraph title: Protein expression and purification ... The E. coli strains BL21(DE3) (Novagen), Rosetta-2(DE3) (Novagen), C41(DE3) (Lucigen, ( )), and C43(DE3) (Lucigen, ( )) were transformed with the TM4-Cx43CT expression plasmid (see above) and then inoculated into 1 L of Luria Bertani medium (LB), enriched minimal media , or ISOGRO media (Sigma-Aldrich).

Plasmid Preparation:

Article Title: A simple vector system to improve performance and utilisation of recombinant antibodies
Article Snippet: .. E. coli BL21 (DE3) and Origami 2™ (DE3) cells and the pET26b(+) plasmid were from Novagen (San Diego, CA). ..

Article Title: Chemical complexity of protein determines optimal E. coli expression host; A comparative study using Erythropoietin, Streptokinase and Tumor Necrosis Factor Receptor
Article Snippet: E. coli BL21 (DE3), E. coli BL21 (DE3) pLys S, E. coli BL21 Rosetta (DE3) pLys S (Novagen,) are IPTG inducible strains in which the T7 RNA Polymerase gene is under the control of a Lac UV5 promoter. .. The expression vector used in this study is pRSET A obtained from Invitrogen Life Technologies, USA. pRSET is a high copy number plasmid with a pUC origin of replication.

Article Title: A Single Codon Optimization Enhances Recombinant Human TNF-α Vaccine Expression in Escherichia coli
Article Snippet: .. The vector pET-22b and E. coli strain BL21 (DE3) were purchased from Novagen (San Diego, CA). .. Isopropyl β -D-1-thiogalactopyranoside (IPTG) was from Sigma-Aldrich (St. Louis, MO).

Article Title: The gaf Fimbrial Gene Cluster of Escherichia coli Expresses a Full-Size and a Truncated Soluble Adhesin Protein
Article Snippet: .. E. coli strain BL21 λDE3 ( ompT lon ) and expression vector pET-22b(+) were from Novagen Inc. (Madison, Wis.). .. The bacteria were cultivated at 37°C in Luria broth ( ) containing the appropriate antibiotics.

Article Title: A novel HPV16 E7-affitoxin for targeted therapy of HPV16-induced human cervical cancer
Article Snippet: .. The pET21a(+) vector and E.coli BL21 (DE3) were purchased from Novagen and ATCC, respectively. .. Reagents The reagents used, including Cell Counting Kit-8 (CCK-8) (Dojindo, Japan), RPMI-1640 (Gibco, USA), fetal bovine serum (FBS) (Gibco, USA), penicillin (Gibco, USA), trypsin-EDTA (Gibco, USA), streptomycin (Sigma Aldrich, Saint Louis, USA), Isopropyl-D-thiogalactopyranoside (IPTG) (Sigma Aldrich, Saint Louis, USA), Ni-NTA agarose (Qiagen Inc., Valencia, CA), and DyLight-755 (Thermo Fisher Scientific, USA), were purchased from commercial sources.

Article Title: Fluorescence Resonance Energy Transfer Assay for High-Throughput Screening of ADAMTS1 Inhibitors
Article Snippet: .. Materials E . coli BL21 (DE3), E. coli DH5a, pET32a(+) vector and his-bind purification kit were purchased from Novagen. .. Heparin sepharose CL-6B column was purchased from Amersham Biosciences.

Article Title: Preparation and Antitumor Activity of CS5931, A Novel Polypeptide from Sea Squirt Ciona Savignyi
Article Snippet: .. Recombinant Expression of CS5931 from E. coli BL21 (DE3) The expression vector, pET28a/CS5931 synthesized by our lab previously [ ], was transfected into E. coli BL21 (DE3) cells (Novagen, Darmstadt, Germany). .. Cells were incubated in low-salt LB medium (containing 100 µg/mL Kanamycin) at 37 °C with shaking at 200 rpm.

Article Title: Identification and molecular characterization of a metagenome-derived L-lysine decarboxylase gene from subtropical soil microorganisms
Article Snippet: .. Plasmid pET-30a(+) (Novagen) and bacterial strain E . coli BL21 (DE3) pLysS (Novagen) were used as the expression vector and host, respectively. ..

Article Title: Purification and Reconstitution of the Connexin43 Carboxyl Terminus attached to the 4th Transmembrane Domain in Detergent Micelles *
Article Snippet: .. The E. coli strains BL21(DE3) (Novagen), Rosetta-2(DE3) (Novagen), C41(DE3) (Lucigen, ( )), and C43(DE3) (Lucigen, ( )) were transformed with the TM4-Cx43CT expression plasmid (see above) and then inoculated into 1 L of Luria Bertani medium (LB), enriched minimal media , or ISOGRO media (Sigma-Aldrich). .. Cultures were incubated at 37°C with continuous agitation.

Concentration Assay:

Article Title: Preparation and Antitumor Activity of CS5931, A Novel Polypeptide from Sea Squirt Ciona Savignyi
Article Snippet: Recombinant Expression of CS5931 from E. coli BL21 (DE3) The expression vector, pET28a/CS5931 synthesized by our lab previously [ ], was transfected into E. coli BL21 (DE3) cells (Novagen, Darmstadt, Germany). .. After the value of OD600 of the culture medium reached around 0.5–0.7, IPTG was added at a final concentration of 0.75 mM and the cells were incubated overnight at 16 °C.

Incubation:

Article Title: Hypoallergenic Der p 1/Der p 2 combination vaccines for immunotherapy of house dust mite allergy
Article Snippet: Expression plasmids containing the Der p 2/1 constructs were transformed into E coli strain BL21 (DE3; Novagen, Merck). .. Cells were lysed by using 3 freeze/thaw cycles (−70°C/+50°C), DNA was degraded by means of incubation with 1 μg of DNase I for 10 minutes at room temperature, and cell debris was removed by means of centrifugation (10,000 g for 30 minutes at 4°C).

Article Title: Preparation and Antitumor Activity of CS5931, A Novel Polypeptide from Sea Squirt Ciona Savignyi
Article Snippet: Recombinant Expression of CS5931 from E. coli BL21 (DE3) The expression vector, pET28a/CS5931 synthesized by our lab previously [ ], was transfected into E. coli BL21 (DE3) cells (Novagen, Darmstadt, Germany). .. Cells were incubated in low-salt LB medium (containing 100 µg/mL Kanamycin) at 37 °C with shaking at 200 rpm.

Article Title: Purification and Reconstitution of the Connexin43 Carboxyl Terminus attached to the 4th Transmembrane Domain in Detergent Micelles *
Article Snippet: The E. coli strains BL21(DE3) (Novagen), Rosetta-2(DE3) (Novagen), C41(DE3) (Lucigen, ( )), and C43(DE3) (Lucigen, ( )) were transformed with the TM4-Cx43CT expression plasmid (see above) and then inoculated into 1 L of Luria Bertani medium (LB), enriched minimal media , or ISOGRO media (Sigma-Aldrich). .. Cultures were incubated at 37°C with continuous agitation.

Cell Culture:

Article Title: A novel HPV16 E7-affitoxin for targeted therapy of HPV16-induced human cervical cancer
Article Snippet: SiHa (ATCC: HTB-35, HPV16 positive, contains about one to two copies of integrated HPV16 genome), CaSki (ATCC: CRL-1550, HPV16 positive, contains about 600 copies of integrated HPV16 genome), HeLa 229 (ATCC: CCL-2.1, HPV18 positive, used as HPV16 negative control cell line), and melanoma tumor A375 (ATCC: CRL-1619, used as HPV negative control cell line) were obtained from the American Type Culture Collection (ATCC, USA) and cultured as previously described . .. The pET21a(+) vector and E.coli BL21 (DE3) were purchased from Novagen and ATCC, respectively.

Activity Assay:

Article Title: A new hybrid bacteriocin, Ent35-MccV, displays antimicrobial activity against pathogenic Gram-positive and Gram-negative bacteria
Article Snippet: .. 2.3 Gene expression and bacteriocins purification for activity determinations Ent35–MccV was expressed from pMA24 in E. coli BL21 [DE3] (pLysS) grown in LB (Sigma) at 37 °C. ..

Negative Control:

Article Title: A novel HPV16 E7-affitoxin for targeted therapy of HPV16-induced human cervical cancer
Article Snippet: SiHa (ATCC: HTB-35, HPV16 positive, contains about one to two copies of integrated HPV16 genome), CaSki (ATCC: CRL-1550, HPV16 positive, contains about 600 copies of integrated HPV16 genome), HeLa 229 (ATCC: CCL-2.1, HPV18 positive, used as HPV16 negative control cell line), and melanoma tumor A375 (ATCC: CRL-1619, used as HPV negative control cell line) were obtained from the American Type Culture Collection (ATCC, USA) and cultured as previously described . .. The pET21a(+) vector and E.coli BL21 (DE3) were purchased from Novagen and ATCC, respectively.

Expressing:

Article Title: A new hybrid bacteriocin, Ent35-MccV, displays antimicrobial activity against pathogenic Gram-positive and Gram-negative bacteria
Article Snippet: .. 2.3 Gene expression and bacteriocins purification for activity determinations Ent35–MccV was expressed from pMA24 in E. coli BL21 [DE3] (pLysS) grown in LB (Sigma) at 37 °C. ..

Article Title: Hypoallergenic Der p 1/Der p 2 combination vaccines for immunotherapy of house dust mite allergy
Article Snippet: .. Expression plasmids containing the Der p 2/1 constructs were transformed into E coli strain BL21 (DE3; Novagen, Merck). .. Protein expression was performed in 250 mL of liquid culture by means of induction with 0.5 mmol/L isopropyl-β-D-1-thiogalactopyranoside at an OD600 of 0.8 for 4 hours at 37°C, and cells were harvested by means of centrifugation at 4000 g for 15 minutes at 4°C.

Article Title: Chemical complexity of protein determines optimal E. coli expression host; A comparative study using Erythropoietin, Streptokinase and Tumor Necrosis Factor Receptor
Article Snippet: The bacterial strains used to study expression were E. coli GJ1158, E. coli BL21 (DE3), E. coli BL21 (DE3) pLys S, E. coli BL21 Rosetta (DE3) pLys S. E. coli GJ1158 , derived from E. coli B strain BL21, is a salt inducible strain with a pro U promoter ( ). .. E. coli BL21 (DE3), E. coli BL21 (DE3) pLys S, E. coli BL21 Rosetta (DE3) pLys S (Novagen,) are IPTG inducible strains in which the T7 RNA Polymerase gene is under the control of a Lac UV5 promoter.

Article Title: Genomic and molecular characterization of a novel quorum sensing molecule in Bacillus licheniformis
Article Snippet: The expression strain [E. coli BL21 (DE3)] and E. coli TOP10 were used for cloning/transformations and were selected on LBA supplemented with ampicilin (100 µgml−1 ). .. E. coli BL21 ComX producer strain was cultivated in M9 minimal salts solution (sigma).

Article Title: The gaf Fimbrial Gene Cluster of Escherichia coli Expresses a Full-Size and a Truncated Soluble Adhesin Protein
Article Snippet: .. E. coli strain BL21 λDE3 ( ompT lon ) and expression vector pET-22b(+) were from Novagen Inc. (Madison, Wis.). .. The bacteria were cultivated at 37°C in Luria broth ( ) containing the appropriate antibiotics.

Article Title: Preparation and Antitumor Activity of CS5931, A Novel Polypeptide from Sea Squirt Ciona Savignyi
Article Snippet: .. Recombinant Expression of CS5931 from E. coli BL21 (DE3) The expression vector, pET28a/CS5931 synthesized by our lab previously [ ], was transfected into E. coli BL21 (DE3) cells (Novagen, Darmstadt, Germany). .. Cells were incubated in low-salt LB medium (containing 100 µg/mL Kanamycin) at 37 °C with shaking at 200 rpm.

Article Title: Purification and Reconstitution of the Connexin43 Carboxyl Terminus attached to the 4th Transmembrane Domain in Detergent Micelles *
Article Snippet: .. The E. coli strains BL21(DE3) (Novagen), Rosetta-2(DE3) (Novagen), C41(DE3) (Lucigen, ( )), and C43(DE3) (Lucigen, ( )) were transformed with the TM4-Cx43CT expression plasmid (see above) and then inoculated into 1 L of Luria Bertani medium (LB), enriched minimal media , or ISOGRO media (Sigma-Aldrich). .. Cultures were incubated at 37°C with continuous agitation.

Transformation Assay:

Article Title: Hypoallergenic Der p 1/Der p 2 combination vaccines for immunotherapy of house dust mite allergy
Article Snippet: .. Expression plasmids containing the Der p 2/1 constructs were transformed into E coli strain BL21 (DE3; Novagen, Merck). .. Protein expression was performed in 250 mL of liquid culture by means of induction with 0.5 mmol/L isopropyl-β-D-1-thiogalactopyranoside at an OD600 of 0.8 for 4 hours at 37°C, and cells were harvested by means of centrifugation at 4000 g for 15 minutes at 4°C.

Article Title: Chemical complexity of protein determines optimal E. coli expression host; A comparative study using Erythropoietin, Streptokinase and Tumor Necrosis Factor Receptor
Article Snippet: 2.1 Bacterial strains and plasmids For initial transformation of the ligation mixtures, the maintenance strain E. coli DH5α was used. .. E. coli BL21 (DE3), E. coli BL21 (DE3) pLys S, E. coli BL21 Rosetta (DE3) pLys S (Novagen,) are IPTG inducible strains in which the T7 RNA Polymerase gene is under the control of a Lac UV5 promoter.

Article Title: Purification and Reconstitution of the Connexin43 Carboxyl Terminus attached to the 4th Transmembrane Domain in Detergent Micelles *
Article Snippet: .. The E. coli strains BL21(DE3) (Novagen), Rosetta-2(DE3) (Novagen), C41(DE3) (Lucigen, ( )), and C43(DE3) (Lucigen, ( )) were transformed with the TM4-Cx43CT expression plasmid (see above) and then inoculated into 1 L of Luria Bertani medium (LB), enriched minimal media , or ISOGRO media (Sigma-Aldrich). .. Cultures were incubated at 37°C with continuous agitation.

Recombinant:

Article Title: Fluorescence Resonance Energy Transfer Assay for High-Throughput Screening of ADAMTS1 Inhibitors
Article Snippet: Materials E . coli BL21 (DE3), E. coli DH5a, pET32a(+) vector and his-bind purification kit were purchased from Novagen. .. Recombinant human ADAMTS1 was purchased from R & D Systems.

Article Title: Preparation and Antitumor Activity of CS5931, A Novel Polypeptide from Sea Squirt Ciona Savignyi
Article Snippet: .. Recombinant Expression of CS5931 from E. coli BL21 (DE3) The expression vector, pET28a/CS5931 synthesized by our lab previously [ ], was transfected into E. coli BL21 (DE3) cells (Novagen, Darmstadt, Germany). .. Cells were incubated in low-salt LB medium (containing 100 µg/mL Kanamycin) at 37 °C with shaking at 200 rpm.

Derivative Assay:

Article Title: Chemical complexity of protein determines optimal E. coli expression host; A comparative study using Erythropoietin, Streptokinase and Tumor Necrosis Factor Receptor
Article Snippet: The bacterial strains used to study expression were E. coli GJ1158, E. coli BL21 (DE3), E. coli BL21 (DE3) pLys S, E. coli BL21 Rosetta (DE3) pLys S. E. coli GJ1158 , derived from E. coli B strain BL21, is a salt inducible strain with a pro U promoter ( ). .. E. coli BL21 (DE3), E. coli BL21 (DE3) pLys S, E. coli BL21 Rosetta (DE3) pLys S (Novagen,) are IPTG inducible strains in which the T7 RNA Polymerase gene is under the control of a Lac UV5 promoter.

Generated:

Article Title: A simple vector system to improve performance and utilisation of recombinant antibodies
Article Snippet: E. coli BL21 (DE3) and Origami 2™ (DE3) cells and the pET26b(+) plasmid were from Novagen (San Diego, CA). .. The other scFvs were generated in house to commercially available antigens purchased from R & D Systems (MN, USA), USBiologicals (MA, USA) and Progen (Heidelberg, Germany).

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  • 94
    Millipore lsrb bl21 ∆ luxs protein
    Binding of <t>LsrB</t> to endogenous AI-2. Purified LsrB (BL21) and LsrB <t>(BL21∆luxS)</t> proteins (5 mg/ml) were incubated for 10 min at 37, 50 and 60 °C to release endogenous AI-2, respectively. After incubation, the LsrB proteins were removed by ultrafiltration (10,000-Da cut-off; EMD Millipore), and the filtered reaction products were tested for AI-2 activity using a V. harveyi BB170 bioassay. The extent of LsrB binding to endogenous AI-2 was evaluated using an AI-2 assay, which showed that recombinant LsrB (BL21) bound to endogenous AI-2 (produced by wild-type strain BL21) and was released from LsrB (BL21) at 50 or 60 °C, respectively ( a ). However, since the luxS mutant BL21∆luxS did not produce endogenous AI-2, no AI-2 could be released from the recombinant LsrB (BL21∆luxS) ( b ). Moreover, the recombinant LuxS protein, which was expressed in strain BL21 (pColdTF-lsrB) as a negative control, also showed no AI-2 binding activity ( c ). AI-2 (10 μM) was used as a positive control
    Lsrb Bl21 ∆ Luxs Protein, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/lsrb bl21 ∆ luxs protein/product/Millipore
    Average 94 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    lsrb bl21 ∆ luxs protein - by Bioz Stars, 2020-04
    94/100 stars
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    93
    Millipore bl21 ∆ luxs
    Construction and identification of luxS mutant strain <t>BL21∆luxS</t>
    Bl21 ∆ Luxs, supplied by Millipore, used in various techniques. Bioz Stars score: 93/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/bl21 ∆ luxs/product/Millipore
    Average 93 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    bl21 ∆ luxs - by Bioz Stars, 2020-04
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    86
    Millipore buffer a bl21 pes2i pellets
    SDS-PAGE analysis of purified protein . Shown are the CaroS2K (A) and CaroS2I (B). Samples were subjected to electrophoresis in 10% polyacrylamide gels, which were stained with Coomassie blue. Lane M, molecular weight standards (kDa); lane 1, cell lysate of E. coli <t>BL21/pET32a;</t> lane 4, cell lysate of BL21/pET30b; lanes 2 and 5, IPTG-induced cell lysates of BL21/pES2kI and <t>BL21/pES2I,</t> respectively; lanes 3 and 6, purified protein obtained after elution. The arrowheads indicate the killing protein of carocin S2K (A) and the immunity protein of carocin S2I (B).
    Buffer A Bl21 Pes2i Pellets, supplied by Millipore, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    buffer a bl21 pes2i pellets - by Bioz Stars, 2020-04
    86/100 stars
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    Image Search Results


    Binding of LsrB to endogenous AI-2. Purified LsrB (BL21) and LsrB (BL21∆luxS) proteins (5 mg/ml) were incubated for 10 min at 37, 50 and 60 °C to release endogenous AI-2, respectively. After incubation, the LsrB proteins were removed by ultrafiltration (10,000-Da cut-off; EMD Millipore), and the filtered reaction products were tested for AI-2 activity using a V. harveyi BB170 bioassay. The extent of LsrB binding to endogenous AI-2 was evaluated using an AI-2 assay, which showed that recombinant LsrB (BL21) bound to endogenous AI-2 (produced by wild-type strain BL21) and was released from LsrB (BL21) at 50 or 60 °C, respectively ( a ). However, since the luxS mutant BL21∆luxS did not produce endogenous AI-2, no AI-2 could be released from the recombinant LsrB (BL21∆luxS) ( b ). Moreover, the recombinant LuxS protein, which was expressed in strain BL21 (pColdTF-lsrB) as a negative control, also showed no AI-2 binding activity ( c ). AI-2 (10 μM) was used as a positive control

    Journal: AMB Express

    Article Title: LsrB-based and temperature-dependent identification of bacterial AI-2 receptor

    doi: 10.1186/s13568-017-0486-y

    Figure Lengend Snippet: Binding of LsrB to endogenous AI-2. Purified LsrB (BL21) and LsrB (BL21∆luxS) proteins (5 mg/ml) were incubated for 10 min at 37, 50 and 60 °C to release endogenous AI-2, respectively. After incubation, the LsrB proteins were removed by ultrafiltration (10,000-Da cut-off; EMD Millipore), and the filtered reaction products were tested for AI-2 activity using a V. harveyi BB170 bioassay. The extent of LsrB binding to endogenous AI-2 was evaluated using an AI-2 assay, which showed that recombinant LsrB (BL21) bound to endogenous AI-2 (produced by wild-type strain BL21) and was released from LsrB (BL21) at 50 or 60 °C, respectively ( a ). However, since the luxS mutant BL21∆luxS did not produce endogenous AI-2, no AI-2 could be released from the recombinant LsrB (BL21∆luxS) ( b ). Moreover, the recombinant LuxS protein, which was expressed in strain BL21 (pColdTF-lsrB) as a negative control, also showed no AI-2 binding activity ( c ). AI-2 (10 μM) was used as a positive control

    Article Snippet: After incubation, the LsrB (BL21∆luxS) protein was removed by ultrafiltration (10,000-Da cut-off, EMD Millipore) and the filtered reaction products were tested for AI-2 activity by investigating the binding activity of LsrB (BL21∆luxS) to exogenous AI-2.

    Techniques: Binding Assay, Purification, Incubation, Activity Assay, Recombinant, Produced, Mutagenesis, Negative Control, Positive Control

    Schematic chart of strategy for binding of LsrB to endogenous AI-2 or exogenous AI-2. The extent of LsrB binding to endogenous AI-2 was evaluated using an AI-2 assay, which showed that recombinant LsrB (BL21) bound to endogenous AI-2 (produced by wild-type strain BL21) and was released from LsrB (BL21) at 50 or 60 °C. However, since the luxS mutant BL21∆luxS did not produce endogenous AI-2, no AI-2 could be released from the recombinant LsrB (BL21∆luxS). The combination of endogenous AI-2 produced by BL21 (DE3), which is known to interfere with the binding of AI-2 to LsrB (BL21), resulted in the loss in the ability of LsrB (BL21) to bind to exogenous AI-2. Hence, the BL21 (DE3) luxS mutant, which was incapable of producing endogenous AI-2, but could bind to exogenous AI-2

    Journal: AMB Express

    Article Title: LsrB-based and temperature-dependent identification of bacterial AI-2 receptor

    doi: 10.1186/s13568-017-0486-y

    Figure Lengend Snippet: Schematic chart of strategy for binding of LsrB to endogenous AI-2 or exogenous AI-2. The extent of LsrB binding to endogenous AI-2 was evaluated using an AI-2 assay, which showed that recombinant LsrB (BL21) bound to endogenous AI-2 (produced by wild-type strain BL21) and was released from LsrB (BL21) at 50 or 60 °C. However, since the luxS mutant BL21∆luxS did not produce endogenous AI-2, no AI-2 could be released from the recombinant LsrB (BL21∆luxS). The combination of endogenous AI-2 produced by BL21 (DE3), which is known to interfere with the binding of AI-2 to LsrB (BL21), resulted in the loss in the ability of LsrB (BL21) to bind to exogenous AI-2. Hence, the BL21 (DE3) luxS mutant, which was incapable of producing endogenous AI-2, but could bind to exogenous AI-2

    Article Snippet: After incubation, the LsrB (BL21∆luxS) protein was removed by ultrafiltration (10,000-Da cut-off, EMD Millipore) and the filtered reaction products were tested for AI-2 activity by investigating the binding activity of LsrB (BL21∆luxS) to exogenous AI-2.

    Techniques: Binding Assay, Recombinant, Produced, Mutagenesis

    SDS-PAGE analysis of total cellular proteins and purified fusion proteins from BL21∆luxS cells. Lane 1 and lane 5: SDS-PAGE analysis of total cellular proteins from BL21∆luxS containing pCold TF (serve as negative control). Lane 2 and lane 6: SDS-PAGE analysis of total cellular proteins containing expression plasmids pCold-TF-lsrB ( a ) and pCold-TF-luxS ( b ), respectively. Lane 3 and lane 7: SDS-PAGE analysis of total precipitation proteins containing expression plasmids pCold-TF-lsrB and pCold-TF-luxS, respectively. Lane 4 and lane 8: elution of the LsrB and LuxS purified fusion protein from the affinity column, respectively

    Journal: AMB Express

    Article Title: LsrB-based and temperature-dependent identification of bacterial AI-2 receptor

    doi: 10.1186/s13568-017-0486-y

    Figure Lengend Snippet: SDS-PAGE analysis of total cellular proteins and purified fusion proteins from BL21∆luxS cells. Lane 1 and lane 5: SDS-PAGE analysis of total cellular proteins from BL21∆luxS containing pCold TF (serve as negative control). Lane 2 and lane 6: SDS-PAGE analysis of total cellular proteins containing expression plasmids pCold-TF-lsrB ( a ) and pCold-TF-luxS ( b ), respectively. Lane 3 and lane 7: SDS-PAGE analysis of total precipitation proteins containing expression plasmids pCold-TF-lsrB and pCold-TF-luxS, respectively. Lane 4 and lane 8: elution of the LsrB and LuxS purified fusion protein from the affinity column, respectively

    Article Snippet: After incubation, the LsrB (BL21∆luxS) protein was removed by ultrafiltration (10,000-Da cut-off, EMD Millipore) and the filtered reaction products were tested for AI-2 activity by investigating the binding activity of LsrB (BL21∆luxS) to exogenous AI-2.

    Techniques: SDS Page, Purification, Negative Control, Expressing, Affinity Column

    Construction and identification of luxS mutant strain BL21∆luxS

    Journal: AMB Express

    Article Title: LsrB-based and temperature-dependent identification of bacterial AI-2 receptor

    doi: 10.1186/s13568-017-0486-y

    Figure Lengend Snippet: Construction and identification of luxS mutant strain BL21∆luxS

    Article Snippet: Cell-free culture fluid (CF) was prepared as follows: E. coli strains BL21 (DE3), BL21∆luxS and DH5a were grown in LB at 37 °C, and then pelleted by centrifugation at 12,000 g at 4 °C for 10 min. Then, the resulting supernatants were filtered through a 0.22-μm filter (EMD Millipore, Bedford, MA, USA) to obtain CF samples.

    Techniques: Mutagenesis

    The AI-2 activity in BL21∆luxS. The wild strain BL21 secretes AI-2-like molecules, and can induce V . harveyi BB170 bioluminescence, whereas no bioluminescence induction was observed for the mutant BL21∆ luxS . V . harveyi BB152 served as

    Journal: AMB Express

    Article Title: LsrB-based and temperature-dependent identification of bacterial AI-2 receptor

    doi: 10.1186/s13568-017-0486-y

    Figure Lengend Snippet: The AI-2 activity in BL21∆luxS. The wild strain BL21 secretes AI-2-like molecules, and can induce V . harveyi BB170 bioluminescence, whereas no bioluminescence induction was observed for the mutant BL21∆ luxS . V . harveyi BB152 served as

    Article Snippet: Cell-free culture fluid (CF) was prepared as follows: E. coli strains BL21 (DE3), BL21∆luxS and DH5a were grown in LB at 37 °C, and then pelleted by centrifugation at 12,000 g at 4 °C for 10 min. Then, the resulting supernatants were filtered through a 0.22-μm filter (EMD Millipore, Bedford, MA, USA) to obtain CF samples.

    Techniques: Activity Assay, Mutagenesis

    SDS-PAGE analysis of total cellular proteins and purified fusion proteins from BL21∆luxS cells. Lane 1 and lane 5: SDS-PAGE analysis of total cellular proteins from BL21∆luxS containing pCold TF (serve as negative control). Lane 2 and

    Journal: AMB Express

    Article Title: LsrB-based and temperature-dependent identification of bacterial AI-2 receptor

    doi: 10.1186/s13568-017-0486-y

    Figure Lengend Snippet: SDS-PAGE analysis of total cellular proteins and purified fusion proteins from BL21∆luxS cells. Lane 1 and lane 5: SDS-PAGE analysis of total cellular proteins from BL21∆luxS containing pCold TF (serve as negative control). Lane 2 and

    Article Snippet: Cell-free culture fluid (CF) was prepared as follows: E. coli strains BL21 (DE3), BL21∆luxS and DH5a were grown in LB at 37 °C, and then pelleted by centrifugation at 12,000 g at 4 °C for 10 min. Then, the resulting supernatants were filtered through a 0.22-μm filter (EMD Millipore, Bedford, MA, USA) to obtain CF samples.

    Techniques: SDS Page, Purification, Negative Control

    Binding of LsrB to endogenous AI-2. Purified LsrB (BL21) and LsrB (BL21∆luxS) proteins (5 mg/ml) were incubated for 10 min at 37, 50 and 60 °C to release endogenous AI-2, respectively. After incubation, the LsrB proteins were removed by ultrafiltration (10,000-Da cut-off; EMD Millipore), and the filtered reaction products were tested for AI-2 activity using a V. harveyi BB170 bioassay. The extent of LsrB binding to endogenous AI-2 was evaluated using an AI-2 assay, which showed that recombinant LsrB (BL21) bound to endogenous AI-2 (produced by wild-type strain BL21) and was released from LsrB (BL21) at 50 or 60 °C, respectively ( a ). However, since the luxS mutant BL21∆luxS did not produce endogenous AI-2, no AI-2 could be released from the recombinant LsrB (BL21∆luxS) ( b ). Moreover, the recombinant LuxS protein, which was expressed in strain BL21 (pColdTF-lsrB) as a negative control, also showed no AI-2 binding activity ( c ). AI-2 (10 μM) was used as a positive control

    Journal: AMB Express

    Article Title: LsrB-based and temperature-dependent identification of bacterial AI-2 receptor

    doi: 10.1186/s13568-017-0486-y

    Figure Lengend Snippet: Binding of LsrB to endogenous AI-2. Purified LsrB (BL21) and LsrB (BL21∆luxS) proteins (5 mg/ml) were incubated for 10 min at 37, 50 and 60 °C to release endogenous AI-2, respectively. After incubation, the LsrB proteins were removed by ultrafiltration (10,000-Da cut-off; EMD Millipore), and the filtered reaction products were tested for AI-2 activity using a V. harveyi BB170 bioassay. The extent of LsrB binding to endogenous AI-2 was evaluated using an AI-2 assay, which showed that recombinant LsrB (BL21) bound to endogenous AI-2 (produced by wild-type strain BL21) and was released from LsrB (BL21) at 50 or 60 °C, respectively ( a ). However, since the luxS mutant BL21∆luxS did not produce endogenous AI-2, no AI-2 could be released from the recombinant LsrB (BL21∆luxS) ( b ). Moreover, the recombinant LuxS protein, which was expressed in strain BL21 (pColdTF-lsrB) as a negative control, also showed no AI-2 binding activity ( c ). AI-2 (10 μM) was used as a positive control

    Article Snippet: Cell-free culture fluid (CF) was prepared as follows: E. coli strains BL21 (DE3), BL21∆luxS and DH5a were grown in LB at 37 °C, and then pelleted by centrifugation at 12,000g at 4 °C for 10 min. Then, the resulting supernatants were filtered through a 0.22-μm filter (EMD Millipore, Bedford, MA, USA) to obtain CF samples.

    Techniques: Binding Assay, Purification, Incubation, Activity Assay, Recombinant, Produced, Mutagenesis, Negative Control, Positive Control

    Schematic chart of strategy for binding of LsrB to endogenous AI-2 or exogenous AI-2. The extent of LsrB binding to endogenous AI-2 was evaluated using an AI-2 assay, which showed that recombinant LsrB (BL21) bound to endogenous AI-2 (produced by wild-type strain BL21) and was released from LsrB (BL21) at 50 or 60 °C. However, since the luxS mutant BL21∆luxS did not produce endogenous AI-2, no AI-2 could be released from the recombinant LsrB (BL21∆luxS). The combination of endogenous AI-2 produced by BL21 (DE3), which is known to interfere with the binding of AI-2 to LsrB (BL21), resulted in the loss in the ability of LsrB (BL21) to bind to exogenous AI-2. Hence, the BL21 (DE3) luxS mutant, which was incapable of producing endogenous AI-2, but could bind to exogenous AI-2

    Journal: AMB Express

    Article Title: LsrB-based and temperature-dependent identification of bacterial AI-2 receptor

    doi: 10.1186/s13568-017-0486-y

    Figure Lengend Snippet: Schematic chart of strategy for binding of LsrB to endogenous AI-2 or exogenous AI-2. The extent of LsrB binding to endogenous AI-2 was evaluated using an AI-2 assay, which showed that recombinant LsrB (BL21) bound to endogenous AI-2 (produced by wild-type strain BL21) and was released from LsrB (BL21) at 50 or 60 °C. However, since the luxS mutant BL21∆luxS did not produce endogenous AI-2, no AI-2 could be released from the recombinant LsrB (BL21∆luxS). The combination of endogenous AI-2 produced by BL21 (DE3), which is known to interfere with the binding of AI-2 to LsrB (BL21), resulted in the loss in the ability of LsrB (BL21) to bind to exogenous AI-2. Hence, the BL21 (DE3) luxS mutant, which was incapable of producing endogenous AI-2, but could bind to exogenous AI-2

    Article Snippet: Cell-free culture fluid (CF) was prepared as follows: E. coli strains BL21 (DE3), BL21∆luxS and DH5a were grown in LB at 37 °C, and then pelleted by centrifugation at 12,000g at 4 °C for 10 min. Then, the resulting supernatants were filtered through a 0.22-μm filter (EMD Millipore, Bedford, MA, USA) to obtain CF samples.

    Techniques: Binding Assay, Recombinant, Produced, Mutagenesis

    The AI-2 activity in BL21∆luxS. The wild strain BL21 secretes AI-2-like molecules, and can induce V . harveyi BB170 bioluminescence, whereas no bioluminescence induction was observed for the mutant BL21∆ luxS . V . harveyi BB152 served as a positive control and E. coli DH5α as a negative control. The figure represents the means of the results from three independent experiments. The error bars indicate standard deviations

    Journal: AMB Express

    Article Title: LsrB-based and temperature-dependent identification of bacterial AI-2 receptor

    doi: 10.1186/s13568-017-0486-y

    Figure Lengend Snippet: The AI-2 activity in BL21∆luxS. The wild strain BL21 secretes AI-2-like molecules, and can induce V . harveyi BB170 bioluminescence, whereas no bioluminescence induction was observed for the mutant BL21∆ luxS . V . harveyi BB152 served as a positive control and E. coli DH5α as a negative control. The figure represents the means of the results from three independent experiments. The error bars indicate standard deviations

    Article Snippet: Cell-free culture fluid (CF) was prepared as follows: E. coli strains BL21 (DE3), BL21∆luxS and DH5a were grown in LB at 37 °C, and then pelleted by centrifugation at 12,000g at 4 °C for 10 min. Then, the resulting supernatants were filtered through a 0.22-μm filter (EMD Millipore, Bedford, MA, USA) to obtain CF samples.

    Techniques: Activity Assay, Mutagenesis, Positive Control, Negative Control

    SDS-PAGE analysis of total cellular proteins and purified fusion proteins from BL21∆luxS cells. Lane 1 and lane 5: SDS-PAGE analysis of total cellular proteins from BL21∆luxS containing pCold TF (serve as negative control). Lane 2 and lane 6: SDS-PAGE analysis of total cellular proteins containing expression plasmids pCold-TF-lsrB ( a ) and pCold-TF-luxS ( b ), respectively. Lane 3 and lane 7: SDS-PAGE analysis of total precipitation proteins containing expression plasmids pCold-TF-lsrB and pCold-TF-luxS, respectively. Lane 4 and lane 8: elution of the LsrB and LuxS purified fusion protein from the affinity column, respectively

    Journal: AMB Express

    Article Title: LsrB-based and temperature-dependent identification of bacterial AI-2 receptor

    doi: 10.1186/s13568-017-0486-y

    Figure Lengend Snippet: SDS-PAGE analysis of total cellular proteins and purified fusion proteins from BL21∆luxS cells. Lane 1 and lane 5: SDS-PAGE analysis of total cellular proteins from BL21∆luxS containing pCold TF (serve as negative control). Lane 2 and lane 6: SDS-PAGE analysis of total cellular proteins containing expression plasmids pCold-TF-lsrB ( a ) and pCold-TF-luxS ( b ), respectively. Lane 3 and lane 7: SDS-PAGE analysis of total precipitation proteins containing expression plasmids pCold-TF-lsrB and pCold-TF-luxS, respectively. Lane 4 and lane 8: elution of the LsrB and LuxS purified fusion protein from the affinity column, respectively

    Article Snippet: Cell-free culture fluid (CF) was prepared as follows: E. coli strains BL21 (DE3), BL21∆luxS and DH5a were grown in LB at 37 °C, and then pelleted by centrifugation at 12,000g at 4 °C for 10 min. Then, the resulting supernatants were filtered through a 0.22-μm filter (EMD Millipore, Bedford, MA, USA) to obtain CF samples.

    Techniques: SDS Page, Purification, Negative Control, Expressing, Affinity Column

    SDS-PAGE analysis of purified protein . Shown are the CaroS2K (A) and CaroS2I (B). Samples were subjected to electrophoresis in 10% polyacrylamide gels, which were stained with Coomassie blue. Lane M, molecular weight standards (kDa); lane 1, cell lysate of E. coli BL21/pET32a; lane 4, cell lysate of BL21/pET30b; lanes 2 and 5, IPTG-induced cell lysates of BL21/pES2kI and BL21/pES2I, respectively; lanes 3 and 6, purified protein obtained after elution. The arrowheads indicate the killing protein of carocin S2K (A) and the immunity protein of carocin S2I (B).

    Journal: BMC Microbiology

    Article Title: Cloning, purification, and functional characterization of Carocin S2, a ribonuclease bacteriocin produced by Pectobacterium carotovorum

    doi: 10.1186/1471-2180-11-99

    Figure Lengend Snippet: SDS-PAGE analysis of purified protein . Shown are the CaroS2K (A) and CaroS2I (B). Samples were subjected to electrophoresis in 10% polyacrylamide gels, which were stained with Coomassie blue. Lane M, molecular weight standards (kDa); lane 1, cell lysate of E. coli BL21/pET32a; lane 4, cell lysate of BL21/pET30b; lanes 2 and 5, IPTG-induced cell lysates of BL21/pES2kI and BL21/pES2I, respectively; lanes 3 and 6, purified protein obtained after elution. The arrowheads indicate the killing protein of carocin S2K (A) and the immunity protein of carocin S2I (B).

    Article Snippet: After purification through a P-100 size-exclusion column (BioRad, USA), the CaroS2K fractions were pooled and concentrated using an Amicon centriprep-50 column (Millipore, USA) and dissolved in buffer A. BL21/pES2I pellets were precipitated by ammonium sulfate (70-100%) and resuspended in buffer A. CaroS2I purification involved a similar chromatographic procedure using the Amicon centriprep-3 column (Millipore, USA).

    Techniques: SDS Page, Purification, Electrophoresis, Staining, Molecular Weight