pmal c2 expression vector  (New England Biolabs)


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    Name:
    pMAL c5X Vector
    Description:
    pMAL c5X Vector 10 ug
    Catalog Number:
    n8108s
    Price:
    131
    Size:
    10 ug
    Category:
    E coli Expression Vectors
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    Structured Review

    New England Biolabs pmal c2 expression vector
    pMAL c5X Vector
    pMAL c5X Vector 10 ug
    https://www.bioz.com/result/pmal c2 expression vector/product/New England Biolabs
    Average 90 stars, based on 157 article reviews
    Price from $9.99 to $1999.99
    pmal c2 expression vector - by Bioz Stars, 2020-01
    90/100 stars

    Images

    1) Product Images from "IDENTIFICATION OF A SPOROZOITE-SPECIFIC ANTIGEN FROM TOXOPLASMA GONDII"

    Article Title: IDENTIFICATION OF A SPOROZOITE-SPECIFIC ANTIGEN FROM TOXOPLASMA GONDII

    Journal: The Journal of parasitology

    doi: 10.1645/GE-2782.1

    Western blots were carried out using human sera and the Factor Xa cleaved-fusion protein purified over a DEAE sepharose column (purified TgERP insert). A, Lane 1, Factor Xa-cleaved, DEAE column purified pMal-c2 vector without the TgERP insert. Lanes 2–7, purified insert, probed with individual sera from Group 1, 6 people from laboratory outbreak resulting from oocyst ingestion. Sera collected 6 weeks post-infection. B, Left, purified insert, probed with pooled sera from 6 Group 1 individuals, 12 weeks post infection; Right; same, probed with pooled sera from 4 chronically infected persons from Group 2. C, purified insert, probed with pooled sera from 6 Group 1 individuals, 5 months post infection D, purified insert, Lanes 1–9, probed with sera of 9 people from stable outbreak (Group 2), Lanes 10–12, probed with sera of 3 IgM positive women from Group 3.
    Figure Legend Snippet: Western blots were carried out using human sera and the Factor Xa cleaved-fusion protein purified over a DEAE sepharose column (purified TgERP insert). A, Lane 1, Factor Xa-cleaved, DEAE column purified pMal-c2 vector without the TgERP insert. Lanes 2–7, purified insert, probed with individual sera from Group 1, 6 people from laboratory outbreak resulting from oocyst ingestion. Sera collected 6 weeks post-infection. B, Left, purified insert, probed with pooled sera from 6 Group 1 individuals, 12 weeks post infection; Right; same, probed with pooled sera from 4 chronically infected persons from Group 2. C, purified insert, probed with pooled sera from 6 Group 1 individuals, 5 months post infection D, purified insert, Lanes 1–9, probed with sera of 9 people from stable outbreak (Group 2), Lanes 10–12, probed with sera of 3 IgM positive women from Group 3.

    Techniques Used: Western Blot, Purification, Plasmid Preparation, Infection

    2) Product Images from "Reovirus Growth in Cell Culture Does Not Require the Full Complement of Viral Proteins: Identification of a ?1s-Null Mutant"

    Article Title: Reovirus Growth in Cell Culture Does Not Require the Full Complement of Viral Proteins: Identification of a ?1s-Null Mutant

    Journal: Journal of Virology

    doi:

    (A) Schematic of full-length and truncated forms of ς1s expressed as fusion proteins with MBP. Sequences of the T3D ς1s ORF were cloned into the pMAL-c2 vector. MBP fusion proteins containing β-galactosidase, full-length ς1s (MBP-ς1s/1–120), or truncations of ς1s (MBP-ς1s/1–84 and MBP-ς1s/1–42) were expressed in E. coli and purified by affinity chromatography using an amylose resin. (B) Immunoblot of MBP-ς1s fusion proteins using MAbs 2F4 and 3E2. The upper gel shows the MBP-β-galactosidase fusion protein as a control and the three MBP-ς1s fusion proteins after electrophoresis in a 10% polyacrylamide gel and staining with Coomassie blue (1 μg of protein per lane). The lower two gels are immunoblots of the same four proteins (20 ng of protein per lane) using MAbs 2F4 and 3E2 (5 μg per ml). Molecular size markers are given in kilodaltons.
    Figure Legend Snippet: (A) Schematic of full-length and truncated forms of ς1s expressed as fusion proteins with MBP. Sequences of the T3D ς1s ORF were cloned into the pMAL-c2 vector. MBP fusion proteins containing β-galactosidase, full-length ς1s (MBP-ς1s/1–120), or truncations of ς1s (MBP-ς1s/1–84 and MBP-ς1s/1–42) were expressed in E. coli and purified by affinity chromatography using an amylose resin. (B) Immunoblot of MBP-ς1s fusion proteins using MAbs 2F4 and 3E2. The upper gel shows the MBP-β-galactosidase fusion protein as a control and the three MBP-ς1s fusion proteins after electrophoresis in a 10% polyacrylamide gel and staining with Coomassie blue (1 μg of protein per lane). The lower two gels are immunoblots of the same four proteins (20 ng of protein per lane) using MAbs 2F4 and 3E2 (5 μg per ml). Molecular size markers are given in kilodaltons.

    Techniques Used: Clone Assay, Plasmid Preparation, Purification, Affinity Chromatography, Electrophoresis, Staining, Western Blot

    3) Product Images from "Two memory associated genes regulated by amyloid precursor protein intracellular domain"

    Article Title: Two memory associated genes regulated by amyloid precursor protein intracellular domain

    Journal: Neural Regeneration Research

    doi: 10.3969/j.issn.1673-5374.2012.05.003

    The maltose-binding protein (MBP)-amyloid precursor protein intracellular domain (AICD) fusion protein was expressed, purified and detected in vitro . (A) Whole-cell extracts from E. coli BL21 transformed with AICD/pMAL-c2 plasmids either noninduced (lane I) or induced by isopropyl-β-D-thiogalactoside (lane II), purified MBP-AICD (lane III) and MBP (lane IV) were separated on 10% sodium dodecyl sulfate-polyacrylamide electrophoresis gels and stained with Coomassie brilliant blue. (B) MBP-AICD (lane I, corresponding to lane III in Figure 1A ) but not MBP (lane II, corresponding to lane IV in Figure 1A ) was recognized by a specific antibody against AICD in western blot analysis.
    Figure Legend Snippet: The maltose-binding protein (MBP)-amyloid precursor protein intracellular domain (AICD) fusion protein was expressed, purified and detected in vitro . (A) Whole-cell extracts from E. coli BL21 transformed with AICD/pMAL-c2 plasmids either noninduced (lane I) or induced by isopropyl-β-D-thiogalactoside (lane II), purified MBP-AICD (lane III) and MBP (lane IV) were separated on 10% sodium dodecyl sulfate-polyacrylamide electrophoresis gels and stained with Coomassie brilliant blue. (B) MBP-AICD (lane I, corresponding to lane III in Figure 1A ) but not MBP (lane II, corresponding to lane IV in Figure 1A ) was recognized by a specific antibody against AICD in western blot analysis.

    Techniques Used: Binding Assay, Purification, In Vitro, Transformation Assay, Electrophoresis, Staining, Western Blot

    4) Product Images from "Arabidopsis Inositol Polyphosphate 6-/3-Kinase Is a Nuclear Protein That Complements a Yeast Mutant Lacking a Functional ArgR-Mcm1 Transcription Complex"

    Article Title: Arabidopsis Inositol Polyphosphate 6-/3-Kinase Is a Nuclear Protein That Complements a Yeast Mutant Lacking a Functional ArgR-Mcm1 Transcription Complex

    Journal: The Plant Cell

    doi: 10.1105/tpc.006676

    Protein Gel Blot Analysis of the MBP-AtIpk2β Fusion Protein. E. coli cells were transformed with either the empty vector pMAL-c2 (lane C) or plasmid pMAL-c2–AtIpk2β (lanes 1 and 2). Protein extracts were obtained from noninduced (−) or IPTG-induced (+) cells. Proteins (45 μg per lane) were detected using an antiserum that recognizes the MBP portion of the proteins. The positions of MBP (42 kD) and the MBP-AtIpk2β fusion protein (75 kD) are indicated by arrows.
    Figure Legend Snippet: Protein Gel Blot Analysis of the MBP-AtIpk2β Fusion Protein. E. coli cells were transformed with either the empty vector pMAL-c2 (lane C) or plasmid pMAL-c2–AtIpk2β (lanes 1 and 2). Protein extracts were obtained from noninduced (−) or IPTG-induced (+) cells. Proteins (45 μg per lane) were detected using an antiserum that recognizes the MBP portion of the proteins. The positions of MBP (42 kD) and the MBP-AtIpk2β fusion protein (75 kD) are indicated by arrows.

    Techniques Used: Western Blot, Transformation Assay, Plasmid Preparation

    5) Product Images from "A DnaB intein in Rhodothermus marinus: Indication of recent intein homing across remotely related organisms"

    Article Title: A DnaB intein in Rhodothermus marinus: Indication of recent intein homing across remotely related organisms

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi:

    Protein splicing of R. marinus DnaB. The R. marinus dnaB gene (complete or partial) was inserted into the expression plasmid vector pMAL-c2 to produce corresponding recombinant fusion proteins and to observe protein splicing. ( A ) Illustration of recombinant fusion proteins. Each fusion protein consists of the maltose-binding protein (MBP), the DnaB intein (solid box), and the DnaB exteins (hatched boxes) of different lengths, and some vector-encoded sequences (open boxs). Calculated molecular masses for the predicted protein products are listed. ( B ) Production and splicing of recombinant DnaB proteins. E. coli cells containing individual recombinant plasmids described above were induced by IPTG to produce the corresponding protein products. Total cellular proteins from induced cells were resolved by electrophoresis on SDS/polyacrylamide gels and visualized by Coomassie blue staining. Lane 1, cells transformed with pMAL and producing a 51-kDa protein, as a control. Lane 2, cells transformed with pMR1, but before IPTG induction. Lanes 3, 4, and 5, cells transformed with pMR1, pMR2, and pMR3, respectively, after IPTG induction. Lanes 6 and 7, same as lanes 3 and 4, respectively, but electrophoresed for a longer period of time. Letters S1, S2, and S3 mark positions of putative spliced proteins produced from pMR1, pMR2, and pMR3, respectively. Letter I marks position of putative excised intein. Letter P marks protein bands that may include precursor proteins and protein splicing intermediates.
    Figure Legend Snippet: Protein splicing of R. marinus DnaB. The R. marinus dnaB gene (complete or partial) was inserted into the expression plasmid vector pMAL-c2 to produce corresponding recombinant fusion proteins and to observe protein splicing. ( A ) Illustration of recombinant fusion proteins. Each fusion protein consists of the maltose-binding protein (MBP), the DnaB intein (solid box), and the DnaB exteins (hatched boxes) of different lengths, and some vector-encoded sequences (open boxs). Calculated molecular masses for the predicted protein products are listed. ( B ) Production and splicing of recombinant DnaB proteins. E. coli cells containing individual recombinant plasmids described above were induced by IPTG to produce the corresponding protein products. Total cellular proteins from induced cells were resolved by electrophoresis on SDS/polyacrylamide gels and visualized by Coomassie blue staining. Lane 1, cells transformed with pMAL and producing a 51-kDa protein, as a control. Lane 2, cells transformed with pMR1, but before IPTG induction. Lanes 3, 4, and 5, cells transformed with pMR1, pMR2, and pMR3, respectively, after IPTG induction. Lanes 6 and 7, same as lanes 3 and 4, respectively, but electrophoresed for a longer period of time. Letters S1, S2, and S3 mark positions of putative spliced proteins produced from pMR1, pMR2, and pMR3, respectively. Letter I marks position of putative excised intein. Letter P marks protein bands that may include precursor proteins and protein splicing intermediates.

    Techniques Used: Expressing, Plasmid Preparation, Recombinant, Binding Assay, Electrophoresis, Staining, Transformation Assay, Produced

    6) Product Images from "Enzymatic Function of the Nor-1 Protein in Aflatoxin Biosynthesis in Aspergillus parasiticus"

    Article Title: Enzymatic Function of the Nor-1 Protein in Aflatoxin Biosynthesis in Aspergillus parasiticus

    Journal: Applied and Environmental Microbiology

    doi:

    Enzyme activity assay of Nor-1c–MBP and Nor-1c proteins. All reactions were conducted at 37°C in the dark. The reaction products were resolved by TLC with benzene-ethyl acetate (7:3) as the developing system. The photograph was taken under white light. The reaction components for each lane are as follows. Lane 1, NA (0.9 mM); lane 2, AVN (0.9 mM); lane 3, NA (0.9 mM) plus NADPH (2.3 mM); lane 4, NA (0.9 mM) plus NADPH (2.3 mM) plus crude cell extract (35 μg) from E. coli DH5α; lane 5, NA (0.9 mM) plus NADPH (2.3 mM) plus crude cell extract (70 μg) from E. coli DH5α containing pMAL-c2; lane 6, NA (0.9 mM) plus NADPH (2.3 mM) plus crude cell extract (70 μg) from E. coli DH5α containing pMN1; lane 7, NA (0.9 mM) plus crude cell extract from (70 μg) E. coli DH5α containing pMN1; lane 8, NA (0.9 mM) plus NADPH (2.3 mM) plus crude cell extract (35 μg) from E. coli DH5α plus purified Nor-1c (35 μg); lane 9, NA (0.9 mM) plus crude cell extract (35 μg) from E. coli DH5α plus purified Nor-1c (35 μg). All crude cell extracts are the 10,000 × g supernatant fraction.
    Figure Legend Snippet: Enzyme activity assay of Nor-1c–MBP and Nor-1c proteins. All reactions were conducted at 37°C in the dark. The reaction products were resolved by TLC with benzene-ethyl acetate (7:3) as the developing system. The photograph was taken under white light. The reaction components for each lane are as follows. Lane 1, NA (0.9 mM); lane 2, AVN (0.9 mM); lane 3, NA (0.9 mM) plus NADPH (2.3 mM); lane 4, NA (0.9 mM) plus NADPH (2.3 mM) plus crude cell extract (35 μg) from E. coli DH5α; lane 5, NA (0.9 mM) plus NADPH (2.3 mM) plus crude cell extract (70 μg) from E. coli DH5α containing pMAL-c2; lane 6, NA (0.9 mM) plus NADPH (2.3 mM) plus crude cell extract (70 μg) from E. coli DH5α containing pMN1; lane 7, NA (0.9 mM) plus crude cell extract from (70 μg) E. coli DH5α containing pMN1; lane 8, NA (0.9 mM) plus NADPH (2.3 mM) plus crude cell extract (35 μg) from E. coli DH5α plus purified Nor-1c (35 μg); lane 9, NA (0.9 mM) plus crude cell extract (35 μg) from E. coli DH5α plus purified Nor-1c (35 μg). All crude cell extracts are the 10,000 × g supernatant fraction.

    Techniques Used: Enzyme Activity Assay, Thin Layer Chromatography, Purification

    Related Articles

    Acetylene Reduction Assay:

    Article Title: Simple In Vitro Assay To Evaluate the Incorporation Efficiency of Ribonucleotide Analog 5′-Triphosphates into RNA by Human Mitochondrial DNA-Dependent RNA Polymerase
    Article Snippet: ATP, UTP, CTP, GTP, 3′-dUTP, 3′-dATP, 3′-dGTP, 3′-dCTP, 2′-dATP, 2′-dCTP, 2′-dGTP, 2′-dTTP, 2′-F-CTP, 2′-F-GTP, 2′-NH2 -GTP, 2′-NH2 -CTP, 2′-F-ATP, 2′-F-UTP, ara-GTP, ara-ATP, 7-deaza-ATP, ara-CTP, ara-UTP, 2′-azido-GTP, and 2′-azido-CTP were purchased as 100 mM solutions from TriLink Biotechnologies (San Diego, CA). .. The pMal-c5X vector was purchased from New England Bioscience (NEB; Ipswich, MA).

    Centrifugation:

    Article Title: The Type Three Secretion System 2-Encoded Regulator EtrB Modulates Enterohemorrhagic Escherichia coli Virulence Gene Expression
    Article Snippet: The EtrB protein was fused with the maltose-binding protein (MBP) using a pMAL-C5x vector (NEB) with the restriction enzymes NcoI and SbfI (NEB) to create pDL02. .. Cells were then pelleted by centrifugation at 4,000 × g for 10 min and resuspended in column buffer (20 mM Tris-HCl, 200 mM NaCl, 1 mM EDTA).

    Amplification:

    Article Title: Metal-bound claMP Tag inhibits proteolytic cleavage
    Article Snippet: .. To create the recombinant DNA, a plasmid containing MBP in the pMAL-C5X vector was obtained (New England Biolabs, Inc.) and the MBP sequence was amplified with the different N-terminal primers (Integrated DNA Technologies) using PCR. .. The primers consisted of a region matching the pET-32Xa/LIC vector shown as underlined, followed by the glycine spacers and cla MP Tag sequence, and lastly a portion matching the N-terminal MBP sequence shown in bold (Gly4, 5′- ATT GAG GGT CGC GGA GGA GGA GGA AAC TGT TGT GGC AAA ATC GAA GAA -3′; Gly3, 5′- GGT ATT GAG GGT CGC GGA GGA GGA AAC TGT TGT GGC AAA ATC GAA GAA -3′; Gly2, 5′- GGT ATT GAG GGT CGC GGA GGA AAC TGT TGT GGC AAA ATC GAA GAA GG -3′; Gly1, 5′- GGT ATT GAG GGT CGC GGA AAC TGT TGT GGC AAA ATC GAA GAA GG -3′; Gly0, 5′- GGT ATT GAG GGT CGC AAC TGT TGT GGC AAA ATC GAA GAA GG -3′; Control, 5′- GGT ATT GAG GGT CGC AAC GCA GCA GGC AAA ATC GAA GAA GG -3′).

    Article Title: Molecular characterization of flavanone 3-hydroxylase gene and flavonoid accumulation in two chemotyped safflower lines in response to methyl jasmonate stimulation
    Article Snippet: .. Expression of recombinant Ct F3H protein, purification, and activity assay The coding region of Ct F3H was amplified from the pMD19 (Takara) into the pMAL-C5x vector (NEB, New England) by using KOD DNA polymerase (Toyobo, Japan) and the primer pair Ct F3H-F/R (forward: 5′-CAAAGAACGTGCcatatgAAACCTAT-3′ with an added NdeI restriction site; reverse: 5′-TCTTAAGCAGATATTTTCTCGATGGGT-3′with an added EcoRI restriction site), which were designed from the sequence of Ct F3H mRNA submitted by our laboratory (GenBank accession no. AEG64806.1). .. The amplified product was sequenced and then digested with restriction enzymes (NEB, New England) to facilitate ligation with the appropriate cloning site of the linearized plasmid pMAL-C5x encoding a maltose-binding protein (MBP).

    Article Title: Rice plants have three homologs of glutathione synthetase genes, one of which, OsGS2, codes for hydroxymethyl‐glutathione synthetase. Rice plants have three homologs of glutathione synthetase genes, one of which, OsGS2, codes for hydroxymethyl‐glutathione synthetase
    Article Snippet: .. The amplified fragment was subcloned into the pMAL‐c5X vector (New England Biolabs) following the manufacturer's instruction. ..

    Article Title: Comparative analyses of ubiquitin-like ATG8 and cysteine protease ATG4 autophagy genes in the plant lineage and cross-kingdom processing of ATG8 by ATG4
    Article Snippet: .. In brief, ORFs of ScATG4 , HsATG4B , AtATG4a (AT2G44140), and SlATG4 (Solyc01g006230) were amplified and cloned into pMal-C2 expression vector (NEW ENGLAND BIOLABS, current version of this vector is pMal-c5X, N8108), resulting in maltose binding protein (MBP)-ATG4. .. For synthetic substrates, ScATG8 , HsLC3A , AtATG8a (AT4G21980), and SlATG8a (Solyc07g064680) ORFs were amplified and cloned into BamHI (NEW ENGLAND BIOLABS, R0136S) and SalI (NEW ENGLAND BIOLABS, R0138S)-restricted pET28-Citrine-ShR plasmid.

    Article Title: Hemoglobin Control of Cell Survival/Death Decision Regulates in Vitro Plant Embryogenesis 1Hemoglobin Control of Cell Survival/Death Decision Regulates in Vitro Plant Embryogenesis 1 [W]Hemoglobin Control of Cell Survival/Death Decision Regulates in Vitro Plant Embryogenesis 1 [W] [OPEN]
    Article Snippet: The amplified product was purified and cloned into the pCR4-blunt TOPO vector (Life Technologies) and confirmed by sequencing. .. An fusion construct was assembled in pMAL-c5X vector (NEB) using an Infusion strategy (Clontech) by linearizing the vector with Xmn I and Eco ).

    Article Title: A PIF1/PIF3-HY5-BBX23 Transcription Factor Cascade Affects Photomorphogenesis 1A PIF1/PIF3-HY5-BBX23 Transcription Factor Cascade Affects Photomorphogenesis 1 [OPEN]
    Article Snippet: The HY5 coding sequence was amplified and ligated into pEASY-Blunt to generate pEASY-HY5. .. The HY5 fragment was released from pEASY-HY5 (cut with Eco RI and Xho I) and cloned into the Eco RI- Xho I sites of the modified pMAL-c5X vector (New England Biolabs), resulting in MBP-HY5.

    Article Title: Resolving Discrepant Findings on ANGPTL8 in β-Cell Proliferation: A Collaborative Approach to Resolving the Betatrophin Controversy
    Article Snippet: Plasmid Expression Vectors cDNA encoding human ANGPTL8 (hBT) protein without the signal peptide (a.a. 22–197) was amplified by PCR. .. Maltose binding protein (MBP) (a.a.1-367) was produced using pMal-c5x vector (New England BioLabs Inc., Ipswich, MA).

    Clone Assay:

    Article Title: Metal-bound claMP Tag inhibits proteolytic cleavage
    Article Snippet: To create the recombinant DNA, a plasmid containing MBP in the pMAL-C5X vector was obtained (New England Biolabs, Inc.) and the MBP sequence was amplified with the different N-terminal primers (Integrated DNA Technologies) using PCR. .. The ligation independent cloning reaction was then transformed into the DH5α Escherichia coli ( E. coli ) cell strain using the standard heat shock procedure.

    Article Title: Molecular characterization of flavanone 3-hydroxylase gene and flavonoid accumulation in two chemotyped safflower lines in response to methyl jasmonate stimulation
    Article Snippet: Expression of recombinant Ct F3H protein, purification, and activity assay The coding region of Ct F3H was amplified from the pMD19 (Takara) into the pMAL-C5x vector (NEB, New England) by using KOD DNA polymerase (Toyobo, Japan) and the primer pair Ct F3H-F/R (forward: 5′-CAAAGAACGTGCcatatgAAACCTAT-3′ with an added NdeI restriction site; reverse: 5′-TCTTAAGCAGATATTTTCTCGATGGGT-3′with an added EcoRI restriction site), which were designed from the sequence of Ct F3H mRNA submitted by our laboratory (GenBank accession no. AEG64806.1). .. The amplified product was sequenced and then digested with restriction enzymes (NEB, New England) to facilitate ligation with the appropriate cloning site of the linearized plasmid pMAL-C5x encoding a maltose-binding protein (MBP).

    Article Title: Simple In Vitro Assay To Evaluate the Incorporation Efficiency of Ribonucleotide Analog 5′-Triphosphates into RNA by Human Mitochondrial DNA-Dependent RNA Polymerase
    Article Snippet: An In-Fusion HD cloning kit and Stellar competent cells were purchased from Clontech (Mountain View, CA). .. The pMal-c5X vector was purchased from New England Bioscience (NEB; Ipswich, MA).

    Article Title: Comparative analyses of ubiquitin-like ATG8 and cysteine protease ATG4 autophagy genes in the plant lineage and cross-kingdom processing of ATG8 by ATG4
    Article Snippet: .. In brief, ORFs of ScATG4 , HsATG4B , AtATG4a (AT2G44140), and SlATG4 (Solyc01g006230) were amplified and cloned into pMal-C2 expression vector (NEW ENGLAND BIOLABS, current version of this vector is pMal-c5X, N8108), resulting in maltose binding protein (MBP)-ATG4. .. For synthetic substrates, ScATG8 , HsLC3A , AtATG8a (AT4G21980), and SlATG8a (Solyc07g064680) ORFs were amplified and cloned into BamHI (NEW ENGLAND BIOLABS, R0136S) and SalI (NEW ENGLAND BIOLABS, R0138S)-restricted pET28-Citrine-ShR plasmid.

    Article Title: Hemoglobin Control of Cell Survival/Death Decision Regulates in Vitro Plant Embryogenesis 1Hemoglobin Control of Cell Survival/Death Decision Regulates in Vitro Plant Embryogenesis 1 [W]Hemoglobin Control of Cell Survival/Death Decision Regulates in Vitro Plant Embryogenesis 1 [W] [OPEN]
    Article Snippet: The amplified product was purified and cloned into the pCR4-blunt TOPO vector (Life Technologies) and confirmed by sequencing. .. An fusion construct was assembled in pMAL-c5X vector (NEB) using an Infusion strategy (Clontech) by linearizing the vector with Xmn I and Eco ).

    Article Title: Post-transcriptional regulation of transcript abundance by a conserved member of the tristetraprolin family in Candida albicans
    Article Snippet: .. Full-length C. albicans ZFS1 (orf19.5334) was cloned using the NotI and BamHI sites of a modified pMAL-c5x vector (New England Biolabs), as described in , and was expressed as a fusion protein with maltose-binding protein linked to the N-terminus of Zfs1 (Zfs1:MBP) by a three alanine linker. .. The recombinant fusion protein was expressed in BL21 (DE3) cells after induction with 0.3 mM IPTG for 16 h at 20°C.

    Article Title: A PIF1/PIF3-HY5-BBX23 Transcription Factor Cascade Affects Photomorphogenesis 1A PIF1/PIF3-HY5-BBX23 Transcription Factor Cascade Affects Photomorphogenesis 1 [OPEN]
    Article Snippet: .. The HY5 fragment was released from pEASY-HY5 (cut with Eco RI and Xho I) and cloned into the Eco RI- Xho I sites of the modified pMAL-c5X vector (New England Biolabs), resulting in MBP-HY5. .. The plasmids of AD-HY5, AD-HY5N, AD-HY5C, AD-PIF1, AD-PIF3, His-HY5, YFPC -HY5, GAL4:LUC, and 35S:GUS were produced as described previously ( ; ; ). pEASY-BBX23 was cut with Xba I and Bam HI to release the BBX23 fragment, which was then ligated into the Xba I- Bam HI site of pCAMBIA-nFlag, giving rise to 35S:Flag-BBX23.

    Construct:

    Article Title: Metal-bound claMP Tag inhibits proteolytic cleavage
    Article Snippet: Six constructs of cla MP-maltose binding protein (MBP) were prepared. .. To create the recombinant DNA, a plasmid containing MBP in the pMAL-C5X vector was obtained (New England Biolabs, Inc.) and the MBP sequence was amplified with the different N-terminal primers (Integrated DNA Technologies) using PCR.

    Article Title: Molecular characterization of flavanone 3-hydroxylase gene and flavonoid accumulation in two chemotyped safflower lines in response to methyl jasmonate stimulation
    Article Snippet: Expression of recombinant Ct F3H protein, purification, and activity assay The coding region of Ct F3H was amplified from the pMD19 (Takara) into the pMAL-C5x vector (NEB, New England) by using KOD DNA polymerase (Toyobo, Japan) and the primer pair Ct F3H-F/R (forward: 5′-CAAAGAACGTGCcatatgAAACCTAT-3′ with an added NdeI restriction site; reverse: 5′-TCTTAAGCAGATATTTTCTCGATGGGT-3′with an added EcoRI restriction site), which were designed from the sequence of Ct F3H mRNA submitted by our laboratory (GenBank accession no. AEG64806.1). .. After validation of its integrity, the construct was introduced into Escherichia coli BL21(DE3)pLysS cells (TransGen Biotech, Beijing) for protein expression.

    Article Title: An Engineered Palette of Metal Ion Quenchable Fluorescent Proteins
    Article Snippet: Plasmids EBFP2 (pBad-EBFP2, #14891), mVenus (mVenus-N1, #27793), and mApple (Myo1E-pmAppleC1, #27698) constructs were from addgene. mEmerald (mEmerald-C1) and mCerulean3 (pmCerulean3-C1) constructs were generous gifts provided by John Hammer (Laboratory of Cell Biology, National Heart Lung and Blood Institute, National Institutes of Health) and Mark Rizzo (School of Medicine, University of Maryland), respectively. mKate2 (pmKate2-C) construct was purchased from Evrogen. .. All these FP genes were subcloned into the pMAL-c5x vector (New England Biolabs).

    Article Title: Hemoglobin Control of Cell Survival/Death Decision Regulates in Vitro Plant Embryogenesis 1Hemoglobin Control of Cell Survival/Death Decision Regulates in Vitro Plant Embryogenesis 1 [W]Hemoglobin Control of Cell Survival/Death Decision Regulates in Vitro Plant Embryogenesis 1 [W] [OPEN]
    Article Snippet: .. An fusion construct was assembled in pMAL-c5X vector (NEB) using an Infusion strategy (Clontech) by linearizing the vector with Xmn I and Eco ). .. A single colony of NEB expression Escherichia coli harboring the pMAL-c5X vector alone or the positive vector containing Zm 4 fusion was used to inoculate an overnight starter culture in 4 mL of Luria-Bertani broth incubated at 37°C with vigorous shaking.

    Article Title: A Rapid Induction Mechanism for Lin28a in Trophic Responses
    Article Snippet: MBP-Lin28a in the pMAL-c5X vector was expressed in BL21 bacteria and purified as described, but rotated with amylose resin (NEB E80215). .. Equal amounts of purified Lin28a protein taken from the same protein aliquot were added to each Sepharose-bound GST construct and rotated at 4°C for 2 hr.

    IA:

    Article Title: Simple In Vitro Assay To Evaluate the Incorporation Efficiency of Ribonucleotide Analog 5′-Triphosphates into RNA by Human Mitochondrial DNA-Dependent RNA Polymerase
    Article Snippet: The pMal-c5X vector was purchased from New England Bioscience (NEB; Ipswich, MA). .. Fluorescently labeled RNA (Cy5.5-RNA) and unlabeled DNA oligonucleotides were chemically synthesized and purified by high-performance liquid chromatography by Integrated DNA Technologies (Coralville, IA).

    Incubation:

    Article Title: Post-transcriptional regulation of transcript abundance by a conserved member of the tristetraprolin family in Candida albicans
    Article Snippet: Full-length C. albicans ZFS1 (orf19.5334) was cloned using the NotI and BamHI sites of a modified pMAL-c5x vector (New England Biolabs), as described in , and was expressed as a fusion protein with maltose-binding protein linked to the N-terminus of Zfs1 (Zfs1:MBP) by a three alanine linker. .. The resulting supernatants were incubated for 1 h at 4°C with amylose resin (New England Biolabs).

    Activity Assay:

    Article Title: Molecular characterization of flavanone 3-hydroxylase gene and flavonoid accumulation in two chemotyped safflower lines in response to methyl jasmonate stimulation
    Article Snippet: .. Expression of recombinant Ct F3H protein, purification, and activity assay The coding region of Ct F3H was amplified from the pMD19 (Takara) into the pMAL-C5x vector (NEB, New England) by using KOD DNA polymerase (Toyobo, Japan) and the primer pair Ct F3H-F/R (forward: 5′-CAAAGAACGTGCcatatgAAACCTAT-3′ with an added NdeI restriction site; reverse: 5′-TCTTAAGCAGATATTTTCTCGATGGGT-3′with an added EcoRI restriction site), which were designed from the sequence of Ct F3H mRNA submitted by our laboratory (GenBank accession no. AEG64806.1). .. The amplified product was sequenced and then digested with restriction enzymes (NEB, New England) to facilitate ligation with the appropriate cloning site of the linearized plasmid pMAL-C5x encoding a maltose-binding protein (MBP).

    Expressing:

    Article Title: Molecular characterization of flavanone 3-hydroxylase gene and flavonoid accumulation in two chemotyped safflower lines in response to methyl jasmonate stimulation
    Article Snippet: .. Expression of recombinant Ct F3H protein, purification, and activity assay The coding region of Ct F3H was amplified from the pMD19 (Takara) into the pMAL-C5x vector (NEB, New England) by using KOD DNA polymerase (Toyobo, Japan) and the primer pair Ct F3H-F/R (forward: 5′-CAAAGAACGTGCcatatgAAACCTAT-3′ with an added NdeI restriction site; reverse: 5′-TCTTAAGCAGATATTTTCTCGATGGGT-3′with an added EcoRI restriction site), which were designed from the sequence of Ct F3H mRNA submitted by our laboratory (GenBank accession no. AEG64806.1). .. The amplified product was sequenced and then digested with restriction enzymes (NEB, New England) to facilitate ligation with the appropriate cloning site of the linearized plasmid pMAL-C5x encoding a maltose-binding protein (MBP).

    Article Title: The Type Three Secretion System 2-Encoded Regulator EtrB Modulates Enterohemorrhagic Escherichia coli Virulence Gene Expression
    Article Snippet: The EtrB protein was fused with the maltose-binding protein (MBP) using a pMAL-C5x vector (NEB) with the restriction enzymes NcoI and SbfI (NEB) to create pDL02. .. Isopropyl-β- d -thiogalactopyranoside (IPTG) was added to a final concentration of 0.3 M, and protein expression was induced overnight at 16°C.

    Article Title: A Novel Matrix Protein, PfY2, Functions as a Crucial Macromolecule during Shell Formation
    Article Snippet: .. Then this PfY2 with a C-His tag was inserted downstream from the malE gene of E . coli ., which encodes maltose-binding protein (MBP), in the pMAL-c5X vector (New England BioLabs Inc., NEB; USA), facilitating the expression of “soluble” fusion protein MBP-tagged PfY2, rPfY2. rPfY2 was purified for further functional analyses. ..

    Article Title: Comparative analyses of ubiquitin-like ATG8 and cysteine protease ATG4 autophagy genes in the plant lineage and cross-kingdom processing of ATG8 by ATG4
    Article Snippet: .. In brief, ORFs of ScATG4 , HsATG4B , AtATG4a (AT2G44140), and SlATG4 (Solyc01g006230) were amplified and cloned into pMal-C2 expression vector (NEW ENGLAND BIOLABS, current version of this vector is pMal-c5X, N8108), resulting in maltose binding protein (MBP)-ATG4. .. For synthetic substrates, ScATG8 , HsLC3A , AtATG8a (AT4G21980), and SlATG8a (Solyc07g064680) ORFs were amplified and cloned into BamHI (NEW ENGLAND BIOLABS, R0136S) and SalI (NEW ENGLAND BIOLABS, R0138S)-restricted pET28-Citrine-ShR plasmid.

    Article Title: Post-transcriptional regulation of transcript abundance by a conserved member of the tristetraprolin family in Candida albicans
    Article Snippet: Paragraph title: Expression and purification of recombinant Zfs1 ... Full-length C. albicans ZFS1 (orf19.5334) was cloned using the NotI and BamHI sites of a modified pMAL-c5x vector (New England Biolabs), as described in , and was expressed as a fusion protein with maltose-binding protein linked to the N-terminus of Zfs1 (Zfs1:MBP) by a three alanine linker.

    Article Title: Resolving Discrepant Findings on ANGPTL8 in β-Cell Proliferation: A Collaborative Approach to Resolving the Betatrophin Controversy
    Article Snippet: Paragraph title: Plasmid Expression Vectors ... Maltose binding protein (MBP) (a.a.1-367) was produced using pMal-c5x vector (New England BioLabs Inc., Ipswich, MA).

    Modification:

    Article Title: Post-transcriptional regulation of transcript abundance by a conserved member of the tristetraprolin family in Candida albicans
    Article Snippet: .. Full-length C. albicans ZFS1 (orf19.5334) was cloned using the NotI and BamHI sites of a modified pMAL-c5x vector (New England Biolabs), as described in , and was expressed as a fusion protein with maltose-binding protein linked to the N-terminus of Zfs1 (Zfs1:MBP) by a three alanine linker. .. The recombinant fusion protein was expressed in BL21 (DE3) cells after induction with 0.3 mM IPTG for 16 h at 20°C.

    Article Title: A PIF1/PIF3-HY5-BBX23 Transcription Factor Cascade Affects Photomorphogenesis 1A PIF1/PIF3-HY5-BBX23 Transcription Factor Cascade Affects Photomorphogenesis 1 [OPEN]
    Article Snippet: .. The HY5 fragment was released from pEASY-HY5 (cut with Eco RI and Xho I) and cloned into the Eco RI- Xho I sites of the modified pMAL-c5X vector (New England Biolabs), resulting in MBP-HY5. .. The plasmids of AD-HY5, AD-HY5N, AD-HY5C, AD-PIF1, AD-PIF3, His-HY5, YFPC -HY5, GAL4:LUC, and 35S:GUS were produced as described previously ( ; ; ). pEASY-BBX23 was cut with Xba I and Bam HI to release the BBX23 fragment, which was then ligated into the Xba I- Bam HI site of pCAMBIA-nFlag, giving rise to 35S:Flag-BBX23.

    Transformation Assay:

    Article Title: Metal-bound claMP Tag inhibits proteolytic cleavage
    Article Snippet: To create the recombinant DNA, a plasmid containing MBP in the pMAL-C5X vector was obtained (New England Biolabs, Inc.) and the MBP sequence was amplified with the different N-terminal primers (Integrated DNA Technologies) using PCR. .. The ligation independent cloning reaction was then transformed into the DH5α Escherichia coli ( E. coli ) cell strain using the standard heat shock procedure.

    Article Title: Comparative analyses of ubiquitin-like ATG8 and cysteine protease ATG4 autophagy genes in the plant lineage and cross-kingdom processing of ATG8 by ATG4
    Article Snippet: In brief, ORFs of ScATG4 , HsATG4B , AtATG4a (AT2G44140), and SlATG4 (Solyc01g006230) were amplified and cloned into pMal-C2 expression vector (NEW ENGLAND BIOLABS, current version of this vector is pMal-c5X, N8108), resulting in maltose binding protein (MBP)-ATG4. .. Plasmids of (HIS)6 -C-ATG8-ShR synthetic substrates and MBP-ATG4 were transformed into E. coli strain BL21 (DE3).

    Article Title: Resolving Discrepant Findings on ANGPTL8 in β-Cell Proliferation: A Collaborative Approach to Resolving the Betatrophin Controversy
    Article Snippet: Maltose binding protein (MBP) (a.a.1-367) was produced using pMal-c5x vector (New England BioLabs Inc., Ipswich, MA). .. The plasmids were transformed into NEB express E .coli ER2523 (New England BioLabs Inc.) for protein expression.

    High Performance Liquid Chromatography:

    Article Title: Simple In Vitro Assay To Evaluate the Incorporation Efficiency of Ribonucleotide Analog 5′-Triphosphates into RNA by Human Mitochondrial DNA-Dependent RNA Polymerase
    Article Snippet: The pMal-c5X vector was purchased from New England Bioscience (NEB; Ipswich, MA). .. Fluorescently labeled RNA (Cy5.5-RNA) and unlabeled DNA oligonucleotides were chemically synthesized and purified by high-performance liquid chromatography by Integrated DNA Technologies (Coralville, IA).

    Ligation:

    Article Title: Metal-bound claMP Tag inhibits proteolytic cleavage
    Article Snippet: To create the recombinant DNA, a plasmid containing MBP in the pMAL-C5X vector was obtained (New England Biolabs, Inc.) and the MBP sequence was amplified with the different N-terminal primers (Integrated DNA Technologies) using PCR. .. The ligation independent cloning reaction was then transformed into the DH5α Escherichia coli ( E. coli ) cell strain using the standard heat shock procedure.

    Article Title: Molecular characterization of flavanone 3-hydroxylase gene and flavonoid accumulation in two chemotyped safflower lines in response to methyl jasmonate stimulation
    Article Snippet: Expression of recombinant Ct F3H protein, purification, and activity assay The coding region of Ct F3H was amplified from the pMD19 (Takara) into the pMAL-C5x vector (NEB, New England) by using KOD DNA polymerase (Toyobo, Japan) and the primer pair Ct F3H-F/R (forward: 5′-CAAAGAACGTGCcatatgAAACCTAT-3′ with an added NdeI restriction site; reverse: 5′-TCTTAAGCAGATATTTTCTCGATGGGT-3′with an added EcoRI restriction site), which were designed from the sequence of Ct F3H mRNA submitted by our laboratory (GenBank accession no. AEG64806.1). .. The amplified product was sequenced and then digested with restriction enzymes (NEB, New England) to facilitate ligation with the appropriate cloning site of the linearized plasmid pMAL-C5x encoding a maltose-binding protein (MBP).

    Protease Inhibitor:

    Article Title: Simple In Vitro Assay To Evaluate the Incorporation Efficiency of Ribonucleotide Analog 5′-Triphosphates into RNA by Human Mitochondrial DNA-Dependent RNA Polymerase
    Article Snippet: LB medium, bovine serum albumin (BSA), NaCl, Triton X-100, glycerol, protease inhibitor cocktail, Tris-HCl (pH 7.5), and HisPur Ni-NTA agarose resin were purchased from Thermo Fisher Scientific (Waltham, MA). .. The pMal-c5X vector was purchased from New England Bioscience (NEB; Ipswich, MA).

    Synthesized:

    Article Title: Simple In Vitro Assay To Evaluate the Incorporation Efficiency of Ribonucleotide Analog 5′-Triphosphates into RNA by Human Mitochondrial DNA-Dependent RNA Polymerase
    Article Snippet: The pMal-c5X vector was purchased from New England Bioscience (NEB; Ipswich, MA). .. Fluorescently labeled RNA (Cy5.5-RNA) and unlabeled DNA oligonucleotides were chemically synthesized and purified by high-performance liquid chromatography by Integrated DNA Technologies (Coralville, IA).

    Generated:

    Article Title: Metal-bound claMP Tag inhibits proteolytic cleavage
    Article Snippet: In addition, a control was generated in which cysteine residues were replaced by alanine to eliminate metal binding (Table ). .. To create the recombinant DNA, a plasmid containing MBP in the pMAL-C5X vector was obtained (New England Biolabs, Inc.) and the MBP sequence was amplified with the different N-terminal primers (Integrated DNA Technologies) using PCR.

    Article Title: An Engineered Palette of Metal Ion Quenchable Fluorescent Proteins
    Article Snippet: All these FP genes were subcloned into the pMAL-c5x vector (New England Biolabs). .. The iq-FP constructs were generated by using the QuikChange II XL Site-Directed Mutagenesis Kit (Agilent Technologies).

    Polymerase Chain Reaction:

    Article Title: Metal-bound claMP Tag inhibits proteolytic cleavage
    Article Snippet: .. To create the recombinant DNA, a plasmid containing MBP in the pMAL-C5X vector was obtained (New England Biolabs, Inc.) and the MBP sequence was amplified with the different N-terminal primers (Integrated DNA Technologies) using PCR. .. The primers consisted of a region matching the pET-32Xa/LIC vector shown as underlined, followed by the glycine spacers and cla MP Tag sequence, and lastly a portion matching the N-terminal MBP sequence shown in bold (Gly4, 5′- ATT GAG GGT CGC GGA GGA GGA GGA AAC TGT TGT GGC AAA ATC GAA GAA -3′; Gly3, 5′- GGT ATT GAG GGT CGC GGA GGA GGA AAC TGT TGT GGC AAA ATC GAA GAA -3′; Gly2, 5′- GGT ATT GAG GGT CGC GGA GGA AAC TGT TGT GGC AAA ATC GAA GAA GG -3′; Gly1, 5′- GGT ATT GAG GGT CGC GGA AAC TGT TGT GGC AAA ATC GAA GAA GG -3′; Gly0, 5′- GGT ATT GAG GGT CGC AAC TGT TGT GGC AAA ATC GAA GAA GG -3′; Control, 5′- GGT ATT GAG GGT CGC AAC GCA GCA GGC AAA ATC GAA GAA GG -3′).

    Article Title: Rice plants have three homologs of glutathione synthetase genes, one of which, OsGS2, codes for hydroxymethyl‐glutathione synthetase. Rice plants have three homologs of glutathione synthetase genes, one of which, OsGS2, codes for hydroxymethyl‐glutathione synthetase
    Article Snippet: 2.6 Preparation and purification of recombinant proteins Full‐length ORF sequences of OsGS1 and OsGS2 were amplified by PCR using Prime Star DNA polymerase (TaKaRa Bio), “Nipponbare” cDNA as the template, and the primer sets, GS1.proF and GS1.proR, or GS2.proF and GS2.proR (Table ). .. The amplified fragment was subcloned into the pMAL‐c5X vector (New England Biolabs) following the manufacturer's instruction.

    Article Title: Resolving Discrepant Findings on ANGPTL8 in β-Cell Proliferation: A Collaborative Approach to Resolving the Betatrophin Controversy
    Article Snippet: Plasmid Expression Vectors cDNA encoding human ANGPTL8 (hBT) protein without the signal peptide (a.a. 22–197) was amplified by PCR. .. Maltose binding protein (MBP) (a.a.1-367) was produced using pMal-c5x vector (New England BioLabs Inc., Ipswich, MA).

    Sonication:

    Article Title: A Rapid Induction Mechanism for Lin28a in Trophic Responses
    Article Snippet: Cultures were centrifuged at 4,000 × g for 20 min at 4°C and lysed in phosphate-buffered saline (PBS) pH = 7.8 with lysozyme (0.5 mg/ml) for 30 min, followed by sonication 5 times with 20 s pulses at 20% amplitude. .. MBP-Lin28a in the pMAL-c5X vector was expressed in BL21 bacteria and purified as described, but rotated with amylose resin (NEB E80215).

    Binding Assay:

    Article Title: Metal-bound claMP Tag inhibits proteolytic cleavage
    Article Snippet: In addition, a control was generated in which cysteine residues were replaced by alanine to eliminate metal binding (Table ). .. To create the recombinant DNA, a plasmid containing MBP in the pMAL-C5X vector was obtained (New England Biolabs, Inc.) and the MBP sequence was amplified with the different N-terminal primers (Integrated DNA Technologies) using PCR.

    Article Title: Soybean GmDREBL Increases Lipid Content in Seeds of Transgenic Arabidopsis
    Article Snippet: .. Gel-shift assay The recombinant protein of maltose binding protein–GmDREBL was expressed in Escherichia coli BL21 using pMAL-c5x vector and purified from cells using Amylose Resin (NEB). ..

    Article Title: Comparative analyses of ubiquitin-like ATG8 and cysteine protease ATG4 autophagy genes in the plant lineage and cross-kingdom processing of ATG8 by ATG4
    Article Snippet: .. In brief, ORFs of ScATG4 , HsATG4B , AtATG4a (AT2G44140), and SlATG4 (Solyc01g006230) were amplified and cloned into pMal-C2 expression vector (NEW ENGLAND BIOLABS, current version of this vector is pMal-c5X, N8108), resulting in maltose binding protein (MBP)-ATG4. .. For synthetic substrates, ScATG8 , HsLC3A , AtATG8a (AT4G21980), and SlATG8a (Solyc07g064680) ORFs were amplified and cloned into BamHI (NEW ENGLAND BIOLABS, R0136S) and SalI (NEW ENGLAND BIOLABS, R0138S)-restricted pET28-Citrine-ShR plasmid.

    Article Title: Resolving Discrepant Findings on ANGPTL8 in β-Cell Proliferation: A Collaborative Approach to Resolving the Betatrophin Controversy
    Article Snippet: .. Maltose binding protein (MBP) (a.a.1-367) was produced using pMal-c5x vector (New England BioLabs Inc., Ipswich, MA). .. Plasmid encoding MBP+hBT protein was a gift from Evotec A.G. (Hamburg, Germany).

    Mutagenesis:

    Article Title: An Engineered Palette of Metal Ion Quenchable Fluorescent Proteins
    Article Snippet: All these FP genes were subcloned into the pMAL-c5x vector (New England Biolabs). .. The iq-FP constructs were generated by using the QuikChange II XL Site-Directed Mutagenesis Kit (Agilent Technologies).

    Isolation:

    Article Title: Hemoglobin Control of Cell Survival/Death Decision Regulates in Vitro Plant Embryogenesis 1Hemoglobin Control of Cell Survival/Death Decision Regulates in Vitro Plant Embryogenesis 1 [W]Hemoglobin Control of Cell Survival/Death Decision Regulates in Vitro Plant Embryogenesis 1 [W] [OPEN]
    Article Snippet: Paragraph title: Isolation of the Maize Embryo-Specific Zm 4 ... An fusion construct was assembled in pMAL-c5X vector (NEB) using an Infusion strategy (Clontech) by linearizing the vector with Xmn I and Eco ).

    Labeling:

    Article Title: Simple In Vitro Assay To Evaluate the Incorporation Efficiency of Ribonucleotide Analog 5′-Triphosphates into RNA by Human Mitochondrial DNA-Dependent RNA Polymerase
    Article Snippet: The pMal-c5X vector was purchased from New England Bioscience (NEB; Ipswich, MA). .. Fluorescently labeled RNA (Cy5.5-RNA) and unlabeled DNA oligonucleotides were chemically synthesized and purified by high-performance liquid chromatography by Integrated DNA Technologies (Coralville, IA).

    Purification:

    Article Title: Metal-bound claMP Tag inhibits proteolytic cleavage
    Article Snippet: To create the recombinant DNA, a plasmid containing MBP in the pMAL-C5X vector was obtained (New England Biolabs, Inc.) and the MBP sequence was amplified with the different N-terminal primers (Integrated DNA Technologies) using PCR. .. The PCR reactions were purified using the QIAquick PCR Purification Kit (Qiagen) and the resulting product was inserted into the pET-32Xa/LIC vector via procedure provided by the manufacturer (Novagen).

    Article Title: The Type Three Secretion System 2-Encoded Regulator EtrB Modulates Enterohemorrhagic Escherichia coli Virulence Gene Expression
    Article Snippet: Paragraph title: Purification of EtrB and QseA. ... The EtrB protein was fused with the maltose-binding protein (MBP) using a pMAL-C5x vector (NEB) with the restriction enzymes NcoI and SbfI (NEB) to create pDL02.

    Article Title: A Novel Matrix Protein, PfY2, Functions as a Crucial Macromolecule during Shell Formation
    Article Snippet: .. Then this PfY2 with a C-His tag was inserted downstream from the malE gene of E . coli ., which encodes maltose-binding protein (MBP), in the pMAL-c5X vector (New England BioLabs Inc., NEB; USA), facilitating the expression of “soluble” fusion protein MBP-tagged PfY2, rPfY2. rPfY2 was purified for further functional analyses. ..

    Article Title: Simple In Vitro Assay To Evaluate the Incorporation Efficiency of Ribonucleotide Analog 5′-Triphosphates into RNA by Human Mitochondrial DNA-Dependent RNA Polymerase
    Article Snippet: The pMal-c5X vector was purchased from New England Bioscience (NEB; Ipswich, MA). .. Fluorescently labeled RNA (Cy5.5-RNA) and unlabeled DNA oligonucleotides were chemically synthesized and purified by high-performance liquid chromatography by Integrated DNA Technologies (Coralville, IA).

    Article Title: Soybean GmDREBL Increases Lipid Content in Seeds of Transgenic Arabidopsis
    Article Snippet: .. Gel-shift assay The recombinant protein of maltose binding protein–GmDREBL was expressed in Escherichia coli BL21 using pMAL-c5x vector and purified from cells using Amylose Resin (NEB). ..

    Article Title: Rice plants have three homologs of glutathione synthetase genes, one of which, OsGS2, codes for hydroxymethyl‐glutathione synthetase. Rice plants have three homologs of glutathione synthetase genes, one of which, OsGS2, codes for hydroxymethyl‐glutathione synthetase
    Article Snippet: Paragraph title: Preparation and purification of recombinant proteins ... The amplified fragment was subcloned into the pMAL‐c5X vector (New England Biolabs) following the manufacturer's instruction.

    Article Title: Comparative analyses of ubiquitin-like ATG8 and cysteine protease ATG4 autophagy genes in the plant lineage and cross-kingdom processing of ATG8 by ATG4
    Article Snippet: Paragraph title: Plasmid construction and purification of ATG4s and ATG8 synthetic substrates ... In brief, ORFs of ScATG4 , HsATG4B , AtATG4a (AT2G44140), and SlATG4 (Solyc01g006230) were amplified and cloned into pMal-C2 expression vector (NEW ENGLAND BIOLABS, current version of this vector is pMal-c5X, N8108), resulting in maltose binding protein (MBP)-ATG4.

    Article Title: Hemoglobin Control of Cell Survival/Death Decision Regulates in Vitro Plant Embryogenesis 1Hemoglobin Control of Cell Survival/Death Decision Regulates in Vitro Plant Embryogenesis 1 [W]Hemoglobin Control of Cell Survival/Death Decision Regulates in Vitro Plant Embryogenesis 1 [W] [OPEN]
    Article Snippet: The amplified product was purified and cloned into the pCR4-blunt TOPO vector (Life Technologies) and confirmed by sequencing. .. An fusion construct was assembled in pMAL-c5X vector (NEB) using an Infusion strategy (Clontech) by linearizing the vector with Xmn I and Eco ).

    Article Title: Post-transcriptional regulation of transcript abundance by a conserved member of the tristetraprolin family in Candida albicans
    Article Snippet: Paragraph title: Expression and purification of recombinant Zfs1 ... Full-length C. albicans ZFS1 (orf19.5334) was cloned using the NotI and BamHI sites of a modified pMAL-c5x vector (New England Biolabs), as described in , and was expressed as a fusion protein with maltose-binding protein linked to the N-terminus of Zfs1 (Zfs1:MBP) by a three alanine linker.

    Article Title: A Rapid Induction Mechanism for Lin28a in Trophic Responses
    Article Snippet: .. MBP-Lin28a in the pMAL-c5X vector was expressed in BL21 bacteria and purified as described, but rotated with amylose resin (NEB E80215). .. Lin28a was cleaved from the amylose-bound MBP tag with factor Xa protease (NEB P8010).

    Protein Purification:

    Article Title: Molecular characterization of flavanone 3-hydroxylase gene and flavonoid accumulation in two chemotyped safflower lines in response to methyl jasmonate stimulation
    Article Snippet: .. Expression of recombinant Ct F3H protein, purification, and activity assay The coding region of Ct F3H was amplified from the pMD19 (Takara) into the pMAL-C5x vector (NEB, New England) by using KOD DNA polymerase (Toyobo, Japan) and the primer pair Ct F3H-F/R (forward: 5′-CAAAGAACGTGCcatatgAAACCTAT-3′ with an added NdeI restriction site; reverse: 5′-TCTTAAGCAGATATTTTCTCGATGGGT-3′with an added EcoRI restriction site), which were designed from the sequence of Ct F3H mRNA submitted by our laboratory (GenBank accession no. AEG64806.1). .. The amplified product was sequenced and then digested with restriction enzymes (NEB, New England) to facilitate ligation with the appropriate cloning site of the linearized plasmid pMAL-C5x encoding a maltose-binding protein (MBP).

    Sequencing:

    Article Title: Metal-bound claMP Tag inhibits proteolytic cleavage
    Article Snippet: .. To create the recombinant DNA, a plasmid containing MBP in the pMAL-C5X vector was obtained (New England Biolabs, Inc.) and the MBP sequence was amplified with the different N-terminal primers (Integrated DNA Technologies) using PCR. .. The primers consisted of a region matching the pET-32Xa/LIC vector shown as underlined, followed by the glycine spacers and cla MP Tag sequence, and lastly a portion matching the N-terminal MBP sequence shown in bold (Gly4, 5′- ATT GAG GGT CGC GGA GGA GGA GGA AAC TGT TGT GGC AAA ATC GAA GAA -3′; Gly3, 5′- GGT ATT GAG GGT CGC GGA GGA GGA AAC TGT TGT GGC AAA ATC GAA GAA -3′; Gly2, 5′- GGT ATT GAG GGT CGC GGA GGA AAC TGT TGT GGC AAA ATC GAA GAA GG -3′; Gly1, 5′- GGT ATT GAG GGT CGC GGA AAC TGT TGT GGC AAA ATC GAA GAA GG -3′; Gly0, 5′- GGT ATT GAG GGT CGC AAC TGT TGT GGC AAA ATC GAA GAA GG -3′; Control, 5′- GGT ATT GAG GGT CGC AAC GCA GCA GGC AAA ATC GAA GAA GG -3′).

    Article Title: Molecular characterization of flavanone 3-hydroxylase gene and flavonoid accumulation in two chemotyped safflower lines in response to methyl jasmonate stimulation
    Article Snippet: .. Expression of recombinant Ct F3H protein, purification, and activity assay The coding region of Ct F3H was amplified from the pMD19 (Takara) into the pMAL-C5x vector (NEB, New England) by using KOD DNA polymerase (Toyobo, Japan) and the primer pair Ct F3H-F/R (forward: 5′-CAAAGAACGTGCcatatgAAACCTAT-3′ with an added NdeI restriction site; reverse: 5′-TCTTAAGCAGATATTTTCTCGATGGGT-3′with an added EcoRI restriction site), which were designed from the sequence of Ct F3H mRNA submitted by our laboratory (GenBank accession no. AEG64806.1). .. The amplified product was sequenced and then digested with restriction enzymes (NEB, New England) to facilitate ligation with the appropriate cloning site of the linearized plasmid pMAL-C5x encoding a maltose-binding protein (MBP).

    Article Title: A Novel Matrix Protein, PfY2, Functions as a Crucial Macromolecule during Shell Formation
    Article Snippet: The DNA sequence of recombinant protein was confirmed by direct sequencing, identical to that of authentic PfY2. .. Then this PfY2 with a C-His tag was inserted downstream from the malE gene of E . coli ., which encodes maltose-binding protein (MBP), in the pMAL-c5X vector (New England BioLabs Inc., NEB; USA), facilitating the expression of “soluble” fusion protein MBP-tagged PfY2, rPfY2. rPfY2 was purified for further functional analyses.

    Article Title: Rice plants have three homologs of glutathione synthetase genes, one of which, OsGS2, codes for hydroxymethyl‐glutathione synthetase. Rice plants have three homologs of glutathione synthetase genes, one of which, OsGS2, codes for hydroxymethyl‐glutathione synthetase
    Article Snippet: The reverse primers contained a recognition sequence for SbfI (GGACGTCC) at the 5′ end. .. The amplified fragment was subcloned into the pMAL‐c5X vector (New England Biolabs) following the manufacturer's instruction.

    Article Title: Hemoglobin Control of Cell Survival/Death Decision Regulates in Vitro Plant Embryogenesis 1Hemoglobin Control of Cell Survival/Death Decision Regulates in Vitro Plant Embryogenesis 1 [W]Hemoglobin Control of Cell Survival/Death Decision Regulates in Vitro Plant Embryogenesis 1 [W] [OPEN]
    Article Snippet: The amplified product was purified and cloned into the pCR4-blunt TOPO vector (Life Technologies) and confirmed by sequencing. .. An fusion construct was assembled in pMAL-c5X vector (NEB) using an Infusion strategy (Clontech) by linearizing the vector with Xmn I and Eco ).

    Article Title: A PIF1/PIF3-HY5-BBX23 Transcription Factor Cascade Affects Photomorphogenesis 1A PIF1/PIF3-HY5-BBX23 Transcription Factor Cascade Affects Photomorphogenesis 1 [OPEN]
    Article Snippet: The HY5 coding sequence was amplified and ligated into pEASY-Blunt to generate pEASY-HY5. .. The HY5 fragment was released from pEASY-HY5 (cut with Eco RI and Xho I) and cloned into the Eco RI- Xho I sites of the modified pMAL-c5X vector (New England Biolabs), resulting in MBP-HY5.

    Positron Emission Tomography:

    Article Title: Metal-bound claMP Tag inhibits proteolytic cleavage
    Article Snippet: To create the recombinant DNA, a plasmid containing MBP in the pMAL-C5X vector was obtained (New England Biolabs, Inc.) and the MBP sequence was amplified with the different N-terminal primers (Integrated DNA Technologies) using PCR. .. The primers consisted of a region matching the pET-32Xa/LIC vector shown as underlined, followed by the glycine spacers and cla MP Tag sequence, and lastly a portion matching the N-terminal MBP sequence shown in bold (Gly4, 5′- ATT GAG GGT CGC GGA GGA GGA GGA AAC TGT TGT GGC AAA ATC GAA GAA -3′; Gly3, 5′- GGT ATT GAG GGT CGC GGA GGA GGA AAC TGT TGT GGC AAA ATC GAA GAA -3′; Gly2, 5′- GGT ATT GAG GGT CGC GGA GGA AAC TGT TGT GGC AAA ATC GAA GAA GG -3′; Gly1, 5′- GGT ATT GAG GGT CGC GGA AAC TGT TGT GGC AAA ATC GAA GAA GG -3′; Gly0, 5′- GGT ATT GAG GGT CGC AAC TGT TGT GGC AAA ATC GAA GAA GG -3′; Control, 5′- GGT ATT GAG GGT CGC AAC GCA GCA GGC AAA ATC GAA GAA GG -3′).

    Article Title: A Novel Matrix Protein, PfY2, Functions as a Crucial Macromolecule during Shell Formation
    Article Snippet: Vector construction The open reading frame (ORF) sequence of target gene PfY2 without signal peptide was introduced into a pET-28a (+) vector (Invitrogen, USA) under the control of a strong T7 promoter, resulting in pET28-rPfY2 with a His6 -tag only at the N-terminus (NH-rPfY2). .. Then this PfY2 with a C-His tag was inserted downstream from the malE gene of E . coli ., which encodes maltose-binding protein (MBP), in the pMAL-c5X vector (New England BioLabs Inc., NEB; USA), facilitating the expression of “soluble” fusion protein MBP-tagged PfY2, rPfY2. rPfY2 was purified for further functional analyses.

    Electrophoretic Mobility Shift Assay:

    Article Title: Soybean GmDREBL Increases Lipid Content in Seeds of Transgenic Arabidopsis
    Article Snippet: .. Gel-shift assay The recombinant protein of maltose binding protein–GmDREBL was expressed in Escherichia coli BL21 using pMAL-c5x vector and purified from cells using Amylose Resin (NEB). ..

    Bradford Protein Assay:

    Article Title: A Rapid Induction Mechanism for Lin28a in Trophic Responses
    Article Snippet: MBP-Lin28a in the pMAL-c5X vector was expressed in BL21 bacteria and purified as described, but rotated with amylose resin (NEB E80215). .. Purified protein concentrations were determined by Bradford Protein Assay, and equivalent amounts of GST-TRBPWT, GST-TRBPSΔD, and GST alone were re-bound to glutathione Sepharose beads by rotation at 4°C for 2 hr.

    Nested PCR:

    Article Title: Rice plants have three homologs of glutathione synthetase genes, one of which, OsGS2, codes for hydroxymethyl‐glutathione synthetase. Rice plants have three homologs of glutathione synthetase genes, one of which, OsGS2, codes for hydroxymethyl‐glutathione synthetase
    Article Snippet: The OsGS3 full‐length sequence was amplified by nested PCR using the following primer sets: step 1, GS3.seqF and GS3.proR; step 2, GS3.proF and GS3.proR. .. The amplified fragment was subcloned into the pMAL‐c5X vector (New England Biolabs) following the manufacturer's instruction.

    Plasmid Preparation:

    Article Title: Metal-bound claMP Tag inhibits proteolytic cleavage
    Article Snippet: .. To create the recombinant DNA, a plasmid containing MBP in the pMAL-C5X vector was obtained (New England Biolabs, Inc.) and the MBP sequence was amplified with the different N-terminal primers (Integrated DNA Technologies) using PCR. .. The primers consisted of a region matching the pET-32Xa/LIC vector shown as underlined, followed by the glycine spacers and cla MP Tag sequence, and lastly a portion matching the N-terminal MBP sequence shown in bold (Gly4, 5′- ATT GAG GGT CGC GGA GGA GGA GGA AAC TGT TGT GGC AAA ATC GAA GAA -3′; Gly3, 5′- GGT ATT GAG GGT CGC GGA GGA GGA AAC TGT TGT GGC AAA ATC GAA GAA -3′; Gly2, 5′- GGT ATT GAG GGT CGC GGA GGA AAC TGT TGT GGC AAA ATC GAA GAA GG -3′; Gly1, 5′- GGT ATT GAG GGT CGC GGA AAC TGT TGT GGC AAA ATC GAA GAA GG -3′; Gly0, 5′- GGT ATT GAG GGT CGC AAC TGT TGT GGC AAA ATC GAA GAA GG -3′; Control, 5′- GGT ATT GAG GGT CGC AAC GCA GCA GGC AAA ATC GAA GAA GG -3′).

    Article Title: Molecular characterization of flavanone 3-hydroxylase gene and flavonoid accumulation in two chemotyped safflower lines in response to methyl jasmonate stimulation
    Article Snippet: .. Expression of recombinant Ct F3H protein, purification, and activity assay The coding region of Ct F3H was amplified from the pMD19 (Takara) into the pMAL-C5x vector (NEB, New England) by using KOD DNA polymerase (Toyobo, Japan) and the primer pair Ct F3H-F/R (forward: 5′-CAAAGAACGTGCcatatgAAACCTAT-3′ with an added NdeI restriction site; reverse: 5′-TCTTAAGCAGATATTTTCTCGATGGGT-3′with an added EcoRI restriction site), which were designed from the sequence of Ct F3H mRNA submitted by our laboratory (GenBank accession no. AEG64806.1). .. The amplified product was sequenced and then digested with restriction enzymes (NEB, New England) to facilitate ligation with the appropriate cloning site of the linearized plasmid pMAL-C5x encoding a maltose-binding protein (MBP).

    Article Title: An Engineered Palette of Metal Ion Quenchable Fluorescent Proteins
    Article Snippet: .. All these FP genes were subcloned into the pMAL-c5x vector (New England Biolabs). .. The iq-FP constructs were generated by using the QuikChange II XL Site-Directed Mutagenesis Kit (Agilent Technologies).

    Article Title: The Type Three Secretion System 2-Encoded Regulator EtrB Modulates Enterohemorrhagic Escherichia coli Virulence Gene Expression
    Article Snippet: .. The EtrB protein was fused with the maltose-binding protein (MBP) using a pMAL-C5x vector (NEB) with the restriction enzymes NcoI and SbfI (NEB) to create pDL02. .. E. coli strain BL21(DE3) containing pDL02 was grown at 37°C in LB broth with glucose (0.2% final concentration) and ampicillin (100 μg/ml) to an OD600 of 0.5.

    Article Title: A Novel Matrix Protein, PfY2, Functions as a Crucial Macromolecule during Shell Formation
    Article Snippet: .. Then this PfY2 with a C-His tag was inserted downstream from the malE gene of E . coli ., which encodes maltose-binding protein (MBP), in the pMAL-c5X vector (New England BioLabs Inc., NEB; USA), facilitating the expression of “soluble” fusion protein MBP-tagged PfY2, rPfY2. rPfY2 was purified for further functional analyses. ..

    Article Title: Simple In Vitro Assay To Evaluate the Incorporation Efficiency of Ribonucleotide Analog 5′-Triphosphates into RNA by Human Mitochondrial DNA-Dependent RNA Polymerase
    Article Snippet: .. The pMal-c5X vector was purchased from New England Bioscience (NEB; Ipswich, MA). .. Fluorescently labeled RNA (Cy5.5-RNA) and unlabeled DNA oligonucleotides were chemically synthesized and purified by high-performance liquid chromatography by Integrated DNA Technologies (Coralville, IA).

    Article Title: Expression and Purification of the Arabidopsis E4 SUMO Ligases PIAL1 and PIAL2
    Article Snippet: .. Rosetta DE3 pLysS strain (Merck KGaA, catalog number: 70956) Vector pMAL-c2X (New England Biolabs, catalog number: N8076S; the current version of the vector sold by NEB is pMAL-c5X, catalog number: N8108S) Peptone (ForMedium, catalog number: PEP03) Yeast extract (ForMedium, catalog number: YEM03) NaCl (Sigma-Aldrich, catalog number: S3014) Chloramphenicol (Sigma-Aldrich, catalog number: C0378) Ampicillin (Sigma-Aldrich, catalog number: A9518) Isopropyl β-D-1-thiogalactopyranoside (IPTG) (GERBU Biotechnik GmbH, catalog number: 1010) KCl (Sigma-Aldrich, catalog number: P5405) Na2 HPO4 (Sigma-Aldrich, catalog number: 71505) KH2 PO4 (Sigma-Aldrich, catalog number: P3786) Aprotinin (Sigma-Aldrich, catalog number: A6279) (solution stored at 4 °C) Leupeptin (Sigma-Aldrich, catalog number: L2884) (stock solution of 1 mg/ml stored in aliquots at -20 °C) Amylose resin (New England Biolabs, catalog number: E8021S) DNase I (Roche Diagnostics, catalog number: 10104159001) Glycerol (GERBU Biotechnik GmbH, catalog number: 2006) Ethylenediamine tetraacetic acid (EDTA) (GERBU Biotechnik GmbH, catalog number: 1034) Tris (GERBU Biotechnik GmbH, catalog number: 1018) Maltose (Merck KGaA, catalog number: 105911) Ultrapure water (18.2 MΩ) Lysogeny broth (LB) (see ) Phosphate buffered saline (PBS) (see ) Column buffer (see ) Elution buffer (see ) .. Orbital shaker at 37 °C Spectrophotometer Cooled centrifuge Bandelin Sonoplus sonicator with an MS 73 tapered probe (BANDELIN electronic GmbH & Co., model: GM70HD) PolyPrep Chromatography Columns (Bio-Rad Laboratories, AbD Serotec® , catalog number: 7311550)

    Article Title: Soybean GmDREBL Increases Lipid Content in Seeds of Transgenic Arabidopsis
    Article Snippet: .. Gel-shift assay The recombinant protein of maltose binding protein–GmDREBL was expressed in Escherichia coli BL21 using pMAL-c5x vector and purified from cells using Amylose Resin (NEB). ..

    Article Title: Rice plants have three homologs of glutathione synthetase genes, one of which, OsGS2, codes for hydroxymethyl‐glutathione synthetase. Rice plants have three homologs of glutathione synthetase genes, one of which, OsGS2, codes for hydroxymethyl‐glutathione synthetase
    Article Snippet: .. The amplified fragment was subcloned into the pMAL‐c5X vector (New England Biolabs) following the manufacturer's instruction. ..

    Article Title: Comparative analyses of ubiquitin-like ATG8 and cysteine protease ATG4 autophagy genes in the plant lineage and cross-kingdom processing of ATG8 by ATG4
    Article Snippet: .. In brief, ORFs of ScATG4 , HsATG4B , AtATG4a (AT2G44140), and SlATG4 (Solyc01g006230) were amplified and cloned into pMal-C2 expression vector (NEW ENGLAND BIOLABS, current version of this vector is pMal-c5X, N8108), resulting in maltose binding protein (MBP)-ATG4. .. For synthetic substrates, ScATG8 , HsLC3A , AtATG8a (AT4G21980), and SlATG8a (Solyc07g064680) ORFs were amplified and cloned into BamHI (NEW ENGLAND BIOLABS, R0136S) and SalI (NEW ENGLAND BIOLABS, R0138S)-restricted pET28-Citrine-ShR plasmid.

    Article Title: Hemoglobin Control of Cell Survival/Death Decision Regulates in Vitro Plant Embryogenesis 1Hemoglobin Control of Cell Survival/Death Decision Regulates in Vitro Plant Embryogenesis 1 [W]Hemoglobin Control of Cell Survival/Death Decision Regulates in Vitro Plant Embryogenesis 1 [W] [OPEN]
    Article Snippet: .. An fusion construct was assembled in pMAL-c5X vector (NEB) using an Infusion strategy (Clontech) by linearizing the vector with Xmn I and Eco ). .. A single colony of NEB expression Escherichia coli harboring the pMAL-c5X vector alone or the positive vector containing Zm 4 fusion was used to inoculate an overnight starter culture in 4 mL of Luria-Bertani broth incubated at 37°C with vigorous shaking.

    Article Title: Post-transcriptional regulation of transcript abundance by a conserved member of the tristetraprolin family in Candida albicans
    Article Snippet: .. Full-length C. albicans ZFS1 (orf19.5334) was cloned using the NotI and BamHI sites of a modified pMAL-c5x vector (New England Biolabs), as described in , and was expressed as a fusion protein with maltose-binding protein linked to the N-terminus of Zfs1 (Zfs1:MBP) by a three alanine linker. .. The recombinant fusion protein was expressed in BL21 (DE3) cells after induction with 0.3 mM IPTG for 16 h at 20°C.

    Article Title: A PIF1/PIF3-HY5-BBX23 Transcription Factor Cascade Affects Photomorphogenesis 1A PIF1/PIF3-HY5-BBX23 Transcription Factor Cascade Affects Photomorphogenesis 1 [OPEN]
    Article Snippet: .. The HY5 fragment was released from pEASY-HY5 (cut with Eco RI and Xho I) and cloned into the Eco RI- Xho I sites of the modified pMAL-c5X vector (New England Biolabs), resulting in MBP-HY5. .. The plasmids of AD-HY5, AD-HY5N, AD-HY5C, AD-PIF1, AD-PIF3, His-HY5, YFPC -HY5, GAL4:LUC, and 35S:GUS were produced as described previously ( ; ; ). pEASY-BBX23 was cut with Xba I and Bam HI to release the BBX23 fragment, which was then ligated into the Xba I- Bam HI site of pCAMBIA-nFlag, giving rise to 35S:Flag-BBX23.

    Article Title: A Rapid Induction Mechanism for Lin28a in Trophic Responses
    Article Snippet: .. MBP-Lin28a in the pMAL-c5X vector was expressed in BL21 bacteria and purified as described, but rotated with amylose resin (NEB E80215). .. Lin28a was cleaved from the amylose-bound MBP tag with factor Xa protease (NEB P8010).

    Article Title: Resolving Discrepant Findings on ANGPTL8 in β-Cell Proliferation: A Collaborative Approach to Resolving the Betatrophin Controversy
    Article Snippet: .. Maltose binding protein (MBP) (a.a.1-367) was produced using pMal-c5x vector (New England BioLabs Inc., Ipswich, MA). .. Plasmid encoding MBP+hBT protein was a gift from Evotec A.G. (Hamburg, Germany).

    Functional Assay:

    Article Title: A Novel Matrix Protein, PfY2, Functions as a Crucial Macromolecule during Shell Formation
    Article Snippet: .. Then this PfY2 with a C-His tag was inserted downstream from the malE gene of E . coli ., which encodes maltose-binding protein (MBP), in the pMAL-c5X vector (New England BioLabs Inc., NEB; USA), facilitating the expression of “soluble” fusion protein MBP-tagged PfY2, rPfY2. rPfY2 was purified for further functional analyses. ..

    Affinity Chromatography:

    Article Title: Rice plants have three homologs of glutathione synthetase genes, one of which, OsGS2, codes for hydroxymethyl‐glutathione synthetase. Rice plants have three homologs of glutathione synthetase genes, one of which, OsGS2, codes for hydroxymethyl‐glutathione synthetase
    Article Snippet: The amplified fragment was subcloned into the pMAL‐c5X vector (New England Biolabs) following the manufacturer's instruction. .. The recombinant maltose‐binding protein (MBP)‐fusion protein was expressed in the Escherichia coli strain NEB Express (ER2523; New England Biolabs) and purified by affinity chromatography using amylose resin (Kellermann & Ferenci, ).

    Produced:

    Article Title: A PIF1/PIF3-HY5-BBX23 Transcription Factor Cascade Affects Photomorphogenesis 1A PIF1/PIF3-HY5-BBX23 Transcription Factor Cascade Affects Photomorphogenesis 1 [OPEN]
    Article Snippet: The HY5 fragment was released from pEASY-HY5 (cut with Eco RI and Xho I) and cloned into the Eco RI- Xho I sites of the modified pMAL-c5X vector (New England Biolabs), resulting in MBP-HY5. .. The plasmids of AD-HY5, AD-HY5N, AD-HY5C, AD-PIF1, AD-PIF3, His-HY5, YFPC -HY5, GAL4:LUC, and 35S:GUS were produced as described previously ( ; ; ). pEASY-BBX23 was cut with Xba I and Bam HI to release the BBX23 fragment, which was then ligated into the Xba I- Bam HI site of pCAMBIA-nFlag, giving rise to 35S:Flag-BBX23.

    Article Title: Resolving Discrepant Findings on ANGPTL8 in β-Cell Proliferation: A Collaborative Approach to Resolving the Betatrophin Controversy
    Article Snippet: .. Maltose binding protein (MBP) (a.a.1-367) was produced using pMal-c5x vector (New England BioLabs Inc., Ipswich, MA). .. Plasmid encoding MBP+hBT protein was a gift from Evotec A.G. (Hamburg, Germany).

    Concentration Assay:

    Article Title: The Type Three Secretion System 2-Encoded Regulator EtrB Modulates Enterohemorrhagic Escherichia coli Virulence Gene Expression
    Article Snippet: The EtrB protein was fused with the maltose-binding protein (MBP) using a pMAL-C5x vector (NEB) with the restriction enzymes NcoI and SbfI (NEB) to create pDL02. .. E. coli strain BL21(DE3) containing pDL02 was grown at 37°C in LB broth with glucose (0.2% final concentration) and ampicillin (100 μg/ml) to an OD600 of 0.5.

    Article Title: A Rapid Induction Mechanism for Lin28a in Trophic Responses
    Article Snippet: RnaseA was added to lysates to a final concentration of 20 μg/ml. .. MBP-Lin28a in the pMAL-c5X vector was expressed in BL21 bacteria and purified as described, but rotated with amylose resin (NEB E80215).

    Recombinant:

    Article Title: Metal-bound claMP Tag inhibits proteolytic cleavage
    Article Snippet: .. To create the recombinant DNA, a plasmid containing MBP in the pMAL-C5X vector was obtained (New England Biolabs, Inc.) and the MBP sequence was amplified with the different N-terminal primers (Integrated DNA Technologies) using PCR. .. The primers consisted of a region matching the pET-32Xa/LIC vector shown as underlined, followed by the glycine spacers and cla MP Tag sequence, and lastly a portion matching the N-terminal MBP sequence shown in bold (Gly4, 5′- ATT GAG GGT CGC GGA GGA GGA GGA AAC TGT TGT GGC AAA ATC GAA GAA -3′; Gly3, 5′- GGT ATT GAG GGT CGC GGA GGA GGA AAC TGT TGT GGC AAA ATC GAA GAA -3′; Gly2, 5′- GGT ATT GAG GGT CGC GGA GGA AAC TGT TGT GGC AAA ATC GAA GAA GG -3′; Gly1, 5′- GGT ATT GAG GGT CGC GGA AAC TGT TGT GGC AAA ATC GAA GAA GG -3′; Gly0, 5′- GGT ATT GAG GGT CGC AAC TGT TGT GGC AAA ATC GAA GAA GG -3′; Control, 5′- GGT ATT GAG GGT CGC AAC GCA GCA GGC AAA ATC GAA GAA GG -3′).

    Article Title: Molecular characterization of flavanone 3-hydroxylase gene and flavonoid accumulation in two chemotyped safflower lines in response to methyl jasmonate stimulation
    Article Snippet: .. Expression of recombinant Ct F3H protein, purification, and activity assay The coding region of Ct F3H was amplified from the pMD19 (Takara) into the pMAL-C5x vector (NEB, New England) by using KOD DNA polymerase (Toyobo, Japan) and the primer pair Ct F3H-F/R (forward: 5′-CAAAGAACGTGCcatatgAAACCTAT-3′ with an added NdeI restriction site; reverse: 5′-TCTTAAGCAGATATTTTCTCGATGGGT-3′with an added EcoRI restriction site), which were designed from the sequence of Ct F3H mRNA submitted by our laboratory (GenBank accession no. AEG64806.1). .. The amplified product was sequenced and then digested with restriction enzymes (NEB, New England) to facilitate ligation with the appropriate cloning site of the linearized plasmid pMAL-C5x encoding a maltose-binding protein (MBP).

    Article Title: A Novel Matrix Protein, PfY2, Functions as a Crucial Macromolecule during Shell Formation
    Article Snippet: The purified recombinant PfY2 with a His6 -tag in this plasmid was used for the synthesis of polyclonal antibody. .. Then this PfY2 with a C-His tag was inserted downstream from the malE gene of E . coli ., which encodes maltose-binding protein (MBP), in the pMAL-c5X vector (New England BioLabs Inc., NEB; USA), facilitating the expression of “soluble” fusion protein MBP-tagged PfY2, rPfY2. rPfY2 was purified for further functional analyses.

    Article Title: Soybean GmDREBL Increases Lipid Content in Seeds of Transgenic Arabidopsis
    Article Snippet: .. Gel-shift assay The recombinant protein of maltose binding protein–GmDREBL was expressed in Escherichia coli BL21 using pMAL-c5x vector and purified from cells using Amylose Resin (NEB). ..

    Article Title: Rice plants have three homologs of glutathione synthetase genes, one of which, OsGS2, codes for hydroxymethyl‐glutathione synthetase. Rice plants have three homologs of glutathione synthetase genes, one of which, OsGS2, codes for hydroxymethyl‐glutathione synthetase
    Article Snippet: Paragraph title: Preparation and purification of recombinant proteins ... The amplified fragment was subcloned into the pMAL‐c5X vector (New England Biolabs) following the manufacturer's instruction.

    Article Title: Post-transcriptional regulation of transcript abundance by a conserved member of the tristetraprolin family in Candida albicans
    Article Snippet: Paragraph title: Expression and purification of recombinant Zfs1 ... Full-length C. albicans ZFS1 (orf19.5334) was cloned using the NotI and BamHI sites of a modified pMAL-c5x vector (New England Biolabs), as described in , and was expressed as a fusion protein with maltose-binding protein linked to the N-terminus of Zfs1 (Zfs1:MBP) by a three alanine linker.

    Article Title: A Rapid Induction Mechanism for Lin28a in Trophic Responses
    Article Snippet: Paragraph title: Purification and pull-down of recombinant bacterial proteins ... MBP-Lin28a in the pMAL-c5X vector was expressed in BL21 bacteria and purified as described, but rotated with amylose resin (NEB E80215).

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    New England Biolabs pmal c2 expression vector
    Pmal C2 Expression Vector, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 90/100, based on 159 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    New England Biolabs pmal c2
    Autocatalytic release of the putative CavV 3CL pro domain from flanking sequences and determination of the C-terminal 3CL pro autoprocessing site. (A) Schematic representation of CavV pp1a/1ab replicase polyproteins and fusion protein constructs used in this experiment. The putative 3CL pro domain is indicated in dark gray, and transmembrane domains are shown in light gray. 3CL pro mut, putative 3CL pro domain containing a Cys-1539-to-Ala change in the expressed sequence. (B and C) Expression analysis of the MBP-pp1a-1343-1720-His 6 (wt) and MBP-pp1a-1343-1720_C1539A-His 6 (mut) fusion proteins. Expression was induced with 1 mM IPTG for 4 h at 18°C. Cell lysates obtained from IPTG-induced and noninduced cells were analyzed in a 14% SDS-polyacrylamide gel (B) or by Western blotting using an MBP-specific monoclonal antibody. The products of the expression control <t>pMAL-c2</t> (MBP-lacZα) and the mutant form of the MBP-3CL pro fusion protein (3CL pro mut) are indicated to the left. Two putative processing products derived from the MBP-3CL pro wt construct are indicated by filled circles. Molecular masses (in kDa) of prestained marker proteins are indicated to the right. (D) Purification of C-terminal (GST-containing) cleavage products produced by 3CL pro -mediated cleavage of the MBP-pp1a-1343-1720-GST fusion protein by glutathione Sepharose chromatography. GST-containing cleavage products eluted in fraction 4 (F4) were subjected to N-terminal sequence analysis by Edman degradation. All three cleavage products were shown to have the N terminus Ser-Ala-Thr-…, suggesting that cleavage occurred at 1700 Q|S 1701 in the CavV pp1a/pp1ab sequence. Molecular masses (in kDa) of marker proteins are indicated to the left. SF, soluble protein fraction; IF, insoluble fraction; FT, column flowthrough fraction; W, wash fraction; F3 to F6, elution fractions 3 to 6.
    Pmal C2, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 90/100, based on 152 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs pbp 2 constructs
    Characterization of individual 345a and 346a insertion mutants of <t>PBP</t> 2
    Pbp 2 Constructs, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 79/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Autocatalytic release of the putative CavV 3CL pro domain from flanking sequences and determination of the C-terminal 3CL pro autoprocessing site. (A) Schematic representation of CavV pp1a/1ab replicase polyproteins and fusion protein constructs used in this experiment. The putative 3CL pro domain is indicated in dark gray, and transmembrane domains are shown in light gray. 3CL pro mut, putative 3CL pro domain containing a Cys-1539-to-Ala change in the expressed sequence. (B and C) Expression analysis of the MBP-pp1a-1343-1720-His 6 (wt) and MBP-pp1a-1343-1720_C1539A-His 6 (mut) fusion proteins. Expression was induced with 1 mM IPTG for 4 h at 18°C. Cell lysates obtained from IPTG-induced and noninduced cells were analyzed in a 14% SDS-polyacrylamide gel (B) or by Western blotting using an MBP-specific monoclonal antibody. The products of the expression control pMAL-c2 (MBP-lacZα) and the mutant form of the MBP-3CL pro fusion protein (3CL pro mut) are indicated to the left. Two putative processing products derived from the MBP-3CL pro wt construct are indicated by filled circles. Molecular masses (in kDa) of prestained marker proteins are indicated to the right. (D) Purification of C-terminal (GST-containing) cleavage products produced by 3CL pro -mediated cleavage of the MBP-pp1a-1343-1720-GST fusion protein by glutathione Sepharose chromatography. GST-containing cleavage products eluted in fraction 4 (F4) were subjected to N-terminal sequence analysis by Edman degradation. All three cleavage products were shown to have the N terminus Ser-Ala-Thr-…, suggesting that cleavage occurred at 1700 Q|S 1701 in the CavV pp1a/pp1ab sequence. Molecular masses (in kDa) of marker proteins are indicated to the left. SF, soluble protein fraction; IF, insoluble fraction; FT, column flowthrough fraction; W, wash fraction; F3 to F6, elution fractions 3 to 6.

    Journal: Journal of Virology

    Article Title: Characterization of an Alphamesonivirus 3C-Like Protease Defines a Special Group of Nidovirus Main Proteases

    doi: 10.1128/JVI.02040-14

    Figure Lengend Snippet: Autocatalytic release of the putative CavV 3CL pro domain from flanking sequences and determination of the C-terminal 3CL pro autoprocessing site. (A) Schematic representation of CavV pp1a/1ab replicase polyproteins and fusion protein constructs used in this experiment. The putative 3CL pro domain is indicated in dark gray, and transmembrane domains are shown in light gray. 3CL pro mut, putative 3CL pro domain containing a Cys-1539-to-Ala change in the expressed sequence. (B and C) Expression analysis of the MBP-pp1a-1343-1720-His 6 (wt) and MBP-pp1a-1343-1720_C1539A-His 6 (mut) fusion proteins. Expression was induced with 1 mM IPTG for 4 h at 18°C. Cell lysates obtained from IPTG-induced and noninduced cells were analyzed in a 14% SDS-polyacrylamide gel (B) or by Western blotting using an MBP-specific monoclonal antibody. The products of the expression control pMAL-c2 (MBP-lacZα) and the mutant form of the MBP-3CL pro fusion protein (3CL pro mut) are indicated to the left. Two putative processing products derived from the MBP-3CL pro wt construct are indicated by filled circles. Molecular masses (in kDa) of prestained marker proteins are indicated to the right. (D) Purification of C-terminal (GST-containing) cleavage products produced by 3CL pro -mediated cleavage of the MBP-pp1a-1343-1720-GST fusion protein by glutathione Sepharose chromatography. GST-containing cleavage products eluted in fraction 4 (F4) were subjected to N-terminal sequence analysis by Edman degradation. All three cleavage products were shown to have the N terminus Ser-Ala-Thr-…, suggesting that cleavage occurred at 1700 Q|S 1701 in the CavV pp1a/pp1ab sequence. Molecular masses (in kDa) of marker proteins are indicated to the left. SF, soluble protein fraction; IF, insoluble fraction; FT, column flowthrough fraction; W, wash fraction; F3 to F6, elution fractions 3 to 6.

    Article Snippet: In a second PCR, the pMAL-c2 (New England BioLabs) backbone was amplified from pMAL-c2 plasmid DNA using oligonucleotides LT-3 and LT-4.

    Techniques: Construct, Sequencing, Expressing, Western Blot, Mutagenesis, Derivative Assay, Marker, Purification, Produced, Chromatography

    Mutation analysis of putative catalytic and substrate-binding residues. (A and B) pMAL-c2-[pp1a-1343-1720-GST] plasmid DNA was used to express CavV pp1a/pp1ab residues 1343 to 1720 in E. coli TB1 cells. Protein expression was induced with 1 mM IPTG (right) for 4 h at 18°C or not induced (left), and total cell lysates were analyzed by SDS-PAGE. (A) Coomassie blue-stained 14% SDS-polyacrylamide gel. (B) Western blot analysis using CavV 3CL pro -specific rabbit antiserum. The MBP-pp1a-1343-1720-GST fusion contained either the 3CL pro wild-type sequence or the same sequence with Ala substitutions for single-amino-acid residues potentially involved in catalysis (Cys-1539, Asp-1465, His-1434, Asp-1467, Glu-1460, and Glu-1470) or specific binding of the P1 residue (His-1554 and Thr-1534). (B) The unprocessed fusion protein precursor (MBP-pp1a-1343-1720-GST), the N-terminally processed cleavage product (3CL pro -pp1a-1700-1720-GST), and the fully processed 3CL pro domain (3CL pro ) are indicated to the right. Molecular masses (in kDa) of marker proteins are indicated to the left.

    Journal: Journal of Virology

    Article Title: Characterization of an Alphamesonivirus 3C-Like Protease Defines a Special Group of Nidovirus Main Proteases

    doi: 10.1128/JVI.02040-14

    Figure Lengend Snippet: Mutation analysis of putative catalytic and substrate-binding residues. (A and B) pMAL-c2-[pp1a-1343-1720-GST] plasmid DNA was used to express CavV pp1a/pp1ab residues 1343 to 1720 in E. coli TB1 cells. Protein expression was induced with 1 mM IPTG (right) for 4 h at 18°C or not induced (left), and total cell lysates were analyzed by SDS-PAGE. (A) Coomassie blue-stained 14% SDS-polyacrylamide gel. (B) Western blot analysis using CavV 3CL pro -specific rabbit antiserum. The MBP-pp1a-1343-1720-GST fusion contained either the 3CL pro wild-type sequence or the same sequence with Ala substitutions for single-amino-acid residues potentially involved in catalysis (Cys-1539, Asp-1465, His-1434, Asp-1467, Glu-1460, and Glu-1470) or specific binding of the P1 residue (His-1554 and Thr-1534). (B) The unprocessed fusion protein precursor (MBP-pp1a-1343-1720-GST), the N-terminally processed cleavage product (3CL pro -pp1a-1700-1720-GST), and the fully processed 3CL pro domain (3CL pro ) are indicated to the right. Molecular masses (in kDa) of marker proteins are indicated to the left.

    Article Snippet: In a second PCR, the pMAL-c2 (New England BioLabs) backbone was amplified from pMAL-c2 plasmid DNA using oligonucleotides LT-3 and LT-4.

    Techniques: Mutagenesis, Binding Assay, Plasmid Preparation, Expressing, SDS Page, Staining, Western Blot, Sequencing, Marker

    Characterization of individual 345a and 346a insertion mutants of PBP 2

    Journal: Biochemistry

    Article Title: A highly conserved interaction involving the middle residue of the SXN active-site motif is crucial for function of Class B penicillin-binding proteins: mutational and computational analysis of PBP 2 from N. gonorrhoeae

    doi: 10.1021/bi2017987

    Figure Lengend Snippet: Characterization of individual 345a and 346a insertion mutants of PBP 2

    Article Snippet: The plasmid for overexpression and purification of PBP 2 constructs (a derivative of pMAL-C2; New England Biolabs, Beverly, MA) was described in earlier publications ( , ).

    Techniques:

    Trypsin sensitivity of wild-type and mutant PBP 2 variants

    Journal: Biochemistry

    Article Title: A highly conserved interaction involving the middle residue of the SXN active-site motif is crucial for function of Class B penicillin-binding proteins: mutational and computational analysis of PBP 2 from N. gonorrhoeae

    doi: 10.1021/bi2017987

    Figure Lengend Snippet: Trypsin sensitivity of wild-type and mutant PBP 2 variants

    Article Snippet: The plasmid for overexpression and purification of PBP 2 constructs (a derivative of pMAL-C2; New England Biolabs, Beverly, MA) was described in earlier publications ( , ).

    Techniques: Mutagenesis