pmal c2x  (New England Biolabs)


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    BamHI 50 000 units
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    r0136l
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    Restriction Enzymes
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    Structured Review

    New England Biolabs pmal c2x
    BamHI
    BamHI 50 000 units
    https://www.bioz.com/result/pmal c2x/product/New England Biolabs
    Average 99 stars, based on 218 article reviews
    Price from $9.99 to $1999.99
    pmal c2x - by Bioz Stars, 2020-08
    99/100 stars

    Images

    1) Product Images from "Overexpression and purification of U24 from human herpesvirus type-6 in E. coli: unconventional use of oxidizing environments with a maltose binding protein-hexahistine dual tag to enhance membrane protein yield"

    Article Title: Overexpression and purification of U24 from human herpesvirus type-6 in E. coli: unconventional use of oxidizing environments with a maltose binding protein-hexahistine dual tag to enhance membrane protein yield

    Journal: Microbial Cell Factories

    doi: 10.1186/1475-2859-10-51

    Examination of cysteine-free mutant U24 expression . The C21SC37S mutant U24 constructs were expressed: pMAL-p2x-U24 in C41 (DE3) and pMAL-c2x-U24 in Origami 2 cells, at 18°C and 37°C. Removal of disulfide bond potential appeared to have no effect on in vivo stability of expressed MBP-6 × His-U24, which exhibited the same expression characteristics as wild-type; a similar loss in mass of the degraded fusion protein is observed at the higher temperature.
    Figure Legend Snippet: Examination of cysteine-free mutant U24 expression . The C21SC37S mutant U24 constructs were expressed: pMAL-p2x-U24 in C41 (DE3) and pMAL-c2x-U24 in Origami 2 cells, at 18°C and 37°C. Removal of disulfide bond potential appeared to have no effect on in vivo stability of expressed MBP-6 × His-U24, which exhibited the same expression characteristics as wild-type; a similar loss in mass of the degraded fusion protein is observed at the higher temperature.

    Techniques Used: Mutagenesis, Expressing, Construct, In Vivo

    U24 codon-optimized gene, amino acid sequence and graphical representation of expressed protein construct . A) BamHI/HindIII cut sites are indicated and used to clone the PCR-amplified duplex DNA into the corresponding sites of pMAL-p2x and pMAL-c2x vectors, from which the MBP-6 × His-U24 fusion protein is expressed. The U24 gene was designed to be preceded by a hexahistidine tag (6 × His) and LVPRGS thrombin cleavage site (indicated by an arrow). Final thrombin-cleaved and purified U24 protein will include an additional two amino acids (Gly-Ser) at the N-terminus. B) Cartoon representation of expressed protein. The difference in constructs is a signal sequence at the N-terminus of the protein expressed by pMAL-p2x-U24, directing expression to the periplasm. The Factor Xa cleavage site is vector-encoded.
    Figure Legend Snippet: U24 codon-optimized gene, amino acid sequence and graphical representation of expressed protein construct . A) BamHI/HindIII cut sites are indicated and used to clone the PCR-amplified duplex DNA into the corresponding sites of pMAL-p2x and pMAL-c2x vectors, from which the MBP-6 × His-U24 fusion protein is expressed. The U24 gene was designed to be preceded by a hexahistidine tag (6 × His) and LVPRGS thrombin cleavage site (indicated by an arrow). Final thrombin-cleaved and purified U24 protein will include an additional two amino acids (Gly-Ser) at the N-terminus. B) Cartoon representation of expressed protein. The difference in constructs is a signal sequence at the N-terminus of the protein expressed by pMAL-p2x-U24, directing expression to the periplasm. The Factor Xa cleavage site is vector-encoded.

    Techniques Used: Sequencing, Construct, Polymerase Chain Reaction, Amplification, Purification, Expressing, Plasmid Preparation

    2) Product Images from "Alternatively spliced exon 5 of the FERM domain of Protein 4.1R encodes a novel binding site for erythrocyte p55 and is critical for membrane targeting in epithelial cells"

    Article Title: Alternatively spliced exon 5 of the FERM domain of Protein 4.1R encodes a novel binding site for erythrocyte p55 and is critical for membrane targeting in epithelial cells

    Journal: Biochimica et biophysica acta

    doi: 10.1016/j.bbamcr.2008.09.012

    Expression of FERM domain constructs and p55 binding assay. (A) Coomassie blue stained gel showing recombinant MBP fusion proteins used for the binding assays. MBP (lanes 1), MBP-FERMΔExon 5 (lane 2), and MBP-FERM (lane 3). Fusion proteins shown in lanes 1–3 were expressed in the cytoplasm of E. coli using the pMal-c2X expression vector. Fusion proteins were also expressed in the bacterial periplasm using the pMal-p2X vector (data not shown). Results were identical with both preparations. (B) Coomassie blue stained gel showing recombinant MBP (lane 1), MBP-exon 5 (lane 2), and MBP-exon 10 (lane 3) used for the binding studies. These recombinant proteins were expressed in the bacterial cytoplasm. Western blot based detection of His-p55 recovered by the MBP-fusion protein conjugated amylose-beads is shown on the right. A monoclonal antibody raised against human erythrocyte p55 was used for Western blotting.
    Figure Legend Snippet: Expression of FERM domain constructs and p55 binding assay. (A) Coomassie blue stained gel showing recombinant MBP fusion proteins used for the binding assays. MBP (lanes 1), MBP-FERMΔExon 5 (lane 2), and MBP-FERM (lane 3). Fusion proteins shown in lanes 1–3 were expressed in the cytoplasm of E. coli using the pMal-c2X expression vector. Fusion proteins were also expressed in the bacterial periplasm using the pMal-p2X vector (data not shown). Results were identical with both preparations. (B) Coomassie blue stained gel showing recombinant MBP (lane 1), MBP-exon 5 (lane 2), and MBP-exon 10 (lane 3) used for the binding studies. These recombinant proteins were expressed in the bacterial cytoplasm. Western blot based detection of His-p55 recovered by the MBP-fusion protein conjugated amylose-beads is shown on the right. A monoclonal antibody raised against human erythrocyte p55 was used for Western blotting.

    Techniques Used: Expressing, Construct, Binding Assay, Staining, Recombinant, Plasmid Preparation, Western Blot

    3) Product Images from "A series of constitutive expression vectors to accurately measure the rate of DNA transposition and correct for auto-inhibition"

    Article Title: A series of constitutive expression vectors to accurately measure the rate of DNA transposition and correct for auto-inhibition

    Journal: bioRxiv

    doi: 10.1101/423012

    SETMAR transposition activity was lost during the same period as Hsmar1 transposase domestication. A/ Phylogenetic tree of anthropoid primates which represents the apparition of mutations in the Hsmar1 domain of SETMAR. All the mutations present in the human SETMAR were tested by papillation assay to determine their effects on Hsmar1 transposition. B/ Representative colonies of pMAL-C2X expressing wild-type (pRC1721) or mutant Hsmar1 transposases (pRC1877-1899).
    Figure Legend Snippet: SETMAR transposition activity was lost during the same period as Hsmar1 transposase domestication. A/ Phylogenetic tree of anthropoid primates which represents the apparition of mutations in the Hsmar1 domain of SETMAR. All the mutations present in the human SETMAR were tested by papillation assay to determine their effects on Hsmar1 transposition. B/ Representative colonies of pMAL-C2X expressing wild-type (pRC1721) or mutant Hsmar1 transposases (pRC1877-1899).

    Techniques Used: Activity Assay, Expressing, Mutagenesis

    4) Product Images from "A series of constitutive expression vectors to accurately measure the rate of DNA transposition and correct for auto-inhibition"

    Article Title: A series of constitutive expression vectors to accurately measure the rate of DNA transposition and correct for auto-inhibition

    Journal: bioRxiv

    doi: 10.1101/423012

    SETMAR transposition activity was lost during the same period as Hsmar1 transposase domestication. A/ Phylogenetic tree of anthropoid primates which represents the apparition of mutations in the Hsmar1 domain of SETMAR. All the mutations present in the human SETMAR were tested by papillation assay to determine their effects on Hsmar1 transposition. B/ Representative colonies of pMAL-C2X expressing wild-type (pRC1721) or mutant Hsmar1 transposases (pRC1877-1899).
    Figure Legend Snippet: SETMAR transposition activity was lost during the same period as Hsmar1 transposase domestication. A/ Phylogenetic tree of anthropoid primates which represents the apparition of mutations in the Hsmar1 domain of SETMAR. All the mutations present in the human SETMAR were tested by papillation assay to determine their effects on Hsmar1 transposition. B/ Representative colonies of pMAL-C2X expressing wild-type (pRC1721) or mutant Hsmar1 transposases (pRC1877-1899).

    Techniques Used: Activity Assay, Expressing, Mutagenesis

    5) Product Images from "A series of constitutive expression vectors to accurately measure the rate of DNA transposition and correct for auto-inhibition"

    Article Title: A series of constitutive expression vectors to accurately measure the rate of DNA transposition and correct for auto-inhibition

    Journal: bioRxiv

    doi: 10.1101/423012

    SETMAR transposition activity was lost during the same period as Hsmar1 transposase domestication. A/ Phylogenetic tree of anthropoid primates which represents the apparition of mutations in the Hsmar1 domain of SETMAR. All the mutations present in the human SETMAR were tested by papillation assay to determine their effects on Hsmar1 transposition. B/ Representative colonies of pMAL-C2X expressing wild-type (pRC1721) or mutant Hsmar1 transposases (pRC1877-1899).
    Figure Legend Snippet: SETMAR transposition activity was lost during the same period as Hsmar1 transposase domestication. A/ Phylogenetic tree of anthropoid primates which represents the apparition of mutations in the Hsmar1 domain of SETMAR. All the mutations present in the human SETMAR were tested by papillation assay to determine their effects on Hsmar1 transposition. B/ Representative colonies of pMAL-C2X expressing wild-type (pRC1721) or mutant Hsmar1 transposases (pRC1877-1899).

    Techniques Used: Activity Assay, Expressing, Mutagenesis

    6) Product Images from "A series of constitutive expression vectors to accurately measure the rate of DNA transposition and correct for auto-inhibition"

    Article Title: A series of constitutive expression vectors to accurately measure the rate of DNA transposition and correct for auto-inhibition

    Journal: bioRxiv

    doi: 10.1101/423012

    SETMAR transposition activity was lost during the same period as Hsmar1 transposase domestication. A/ Phylogenetic tree of anthropoid primates which represents the apparition of mutations in the Hsmar1 domain of SETMAR. All the mutations present in the human SETMAR were tested by papillation assay to determine their effects on Hsmar1 transposition. B/ Representative colonies of pMAL-C2X expressing wild-type (pRC1721) or mutant Hsmar1 transposases (pRC1877-1899).
    Figure Legend Snippet: SETMAR transposition activity was lost during the same period as Hsmar1 transposase domestication. A/ Phylogenetic tree of anthropoid primates which represents the apparition of mutations in the Hsmar1 domain of SETMAR. All the mutations present in the human SETMAR were tested by papillation assay to determine their effects on Hsmar1 transposition. B/ Representative colonies of pMAL-C2X expressing wild-type (pRC1721) or mutant Hsmar1 transposases (pRC1877-1899).

    Techniques Used: Activity Assay, Expressing, Mutagenesis

    7) Product Images from "Compensating for over-production inhibition of the Hsmar1 transposon in Escherichia coli using a series of constitutive promoters"

    Article Title: Compensating for over-production inhibition of the Hsmar1 transposon in Escherichia coli using a series of constitutive promoters

    Journal: Mobile DNA

    doi: 10.1186/s13100-020-0200-5

    SETMAR transposition activity was lost during the same period as Hsmar1 transposase domestication. a . Phylogenetic tree of anthropoid primates which represents the emergence of mutations in the Hsmar1 domain of SETMAR. All the mutations present in the human SETMAR were tested by papillation assay to determine their effects on Hsmar1 transposition. The colour code used for the effect of the mutation on the number of papillae per colony is based on Fig. 2c. b . Representative colonies of pMAL-C2X expressing wild-type (pRC1721) or mutant Hsmar1 transposases (pRC1877–1899). The papillation assays were performed in presence of 0.01% lactose and no IPTG. c . Quantification of the number of papillae per colony from single colonies. Average ± standard deviation of six representative colonies from the same biological replicate
    Figure Legend Snippet: SETMAR transposition activity was lost during the same period as Hsmar1 transposase domestication. a . Phylogenetic tree of anthropoid primates which represents the emergence of mutations in the Hsmar1 domain of SETMAR. All the mutations present in the human SETMAR were tested by papillation assay to determine their effects on Hsmar1 transposition. The colour code used for the effect of the mutation on the number of papillae per colony is based on Fig. 2c. b . Representative colonies of pMAL-C2X expressing wild-type (pRC1721) or mutant Hsmar1 transposases (pRC1877–1899). The papillation assays were performed in presence of 0.01% lactose and no IPTG. c . Quantification of the number of papillae per colony from single colonies. Average ± standard deviation of six representative colonies from the same biological replicate

    Techniques Used: Activity Assay, Mutagenesis, Expressing, Standard Deviation

    8) Product Images from "The RNA binding of protein A from Wuhan nodavirus is mediated by mitochondrial membrane lipids"

    Article Title: The RNA binding of protein A from Wuhan nodavirus is mediated by mitochondrial membrane lipids

    Journal: Virology

    doi: 10.1016/j.virol.2014.05.022

    The binding preference of recombinant protein A to RNA1. (A) SDS-PAGE analysis of purified recombinant protein A from E. coli . Protein A ORF was cloned into pMAL-c2X and expressed as C-terminal fusion proteins with MBP (MBP-protA) as described previously ( Qiu et al., 2014 ). Lane 1, Marker; lane 2, MBP protein alone; lane 3, MBP-protA. (B) Gel mobility shift assay showing interactions between MBP-protA and RNA1. The in vitro transcribed DIG-labeled RNA1 (50–118) was separately incubated with bovine serum albumin (BSA, lane 2), MBP alone (lane 3), boiled MBP-protA (lane 4) and MBP-protA (lane 5) (3 μM each), in a binding buffer at 27 °C for 30 min and then analyzed in 1% agarose gel. Gel was transferred to Hybond N nylon membranes via capillary transfer and then the membranes were incubated with anti-DIG antibody conjugated with alkaline phosphatase, exposed to film. The unbound, free RNA1 (50–118) probe and the shift (bound) RNA-protein complex are marked on the right. (C) Unlabeled competitor RNAs at increasing concentrations (in 1-, 10-, 60-fold excess) were added to the mixture containing the DIG-labeled RNA1 (50–118) and 3 μM MBP-protA, and the bound complexes were analyzed in a gel mobility shift assay. The tRNA was from yeast. (D) Gel mobility shift assay showing interactions between MBP-protA and RNA2. The in vitro transcribed DIG-labeled RNA2 (123–164) was incubated with MBP-protA (lane 2) and in a binding buffer at 27 °C for 30 min and then analyzed in 1% agarose gel. Unlabeled competitor RNAs at increasing concentrations (in 5-, 50-, 100-fold excess) were added to the mixture containing the DIG-labeled RNA2 (123–164) and 3 μM MBP-protA, and the bound complexes were analyzed in a gel mobility shift assay. (E) Cooperative binding of MBP-protA to RNA1 (50–118) . Gel mobility shift assays were performed using increasing molar concentrations of MBP-protA incubated with 20nM RNA1 (50–118) probe. The molar concentrations of MBP-protA (0.1–6 μM) are indicated above each lane. (F) The plot of the percent of RNA bound versus molar concentration of MBP-protA. (G) The Hill coefficients of the RNA binding of protein A based on Fig. 2 E at low and high protein concentrations are indicated.
    Figure Legend Snippet: The binding preference of recombinant protein A to RNA1. (A) SDS-PAGE analysis of purified recombinant protein A from E. coli . Protein A ORF was cloned into pMAL-c2X and expressed as C-terminal fusion proteins with MBP (MBP-protA) as described previously ( Qiu et al., 2014 ). Lane 1, Marker; lane 2, MBP protein alone; lane 3, MBP-protA. (B) Gel mobility shift assay showing interactions between MBP-protA and RNA1. The in vitro transcribed DIG-labeled RNA1 (50–118) was separately incubated with bovine serum albumin (BSA, lane 2), MBP alone (lane 3), boiled MBP-protA (lane 4) and MBP-protA (lane 5) (3 μM each), in a binding buffer at 27 °C for 30 min and then analyzed in 1% agarose gel. Gel was transferred to Hybond N nylon membranes via capillary transfer and then the membranes were incubated with anti-DIG antibody conjugated with alkaline phosphatase, exposed to film. The unbound, free RNA1 (50–118) probe and the shift (bound) RNA-protein complex are marked on the right. (C) Unlabeled competitor RNAs at increasing concentrations (in 1-, 10-, 60-fold excess) were added to the mixture containing the DIG-labeled RNA1 (50–118) and 3 μM MBP-protA, and the bound complexes were analyzed in a gel mobility shift assay. The tRNA was from yeast. (D) Gel mobility shift assay showing interactions between MBP-protA and RNA2. The in vitro transcribed DIG-labeled RNA2 (123–164) was incubated with MBP-protA (lane 2) and in a binding buffer at 27 °C for 30 min and then analyzed in 1% agarose gel. Unlabeled competitor RNAs at increasing concentrations (in 5-, 50-, 100-fold excess) were added to the mixture containing the DIG-labeled RNA2 (123–164) and 3 μM MBP-protA, and the bound complexes were analyzed in a gel mobility shift assay. (E) Cooperative binding of MBP-protA to RNA1 (50–118) . Gel mobility shift assays were performed using increasing molar concentrations of MBP-protA incubated with 20nM RNA1 (50–118) probe. The molar concentrations of MBP-protA (0.1–6 μM) are indicated above each lane. (F) The plot of the percent of RNA bound versus molar concentration of MBP-protA. (G) The Hill coefficients of the RNA binding of protein A based on Fig. 2 E at low and high protein concentrations are indicated.

    Techniques Used: Binding Assay, Recombinant, SDS Page, Purification, Clone Assay, Marker, Mobility Shift, In Vitro, Labeling, Incubation, Agarose Gel Electrophoresis, Concentration Assay, RNA Binding Assay

    9) Product Images from "Fusion expression of Helicobacter pylori neutrophil-activating protein in E.coli"

    Article Title: Fusion expression of Helicobacter pylori neutrophil-activating protein in E.coli

    Journal: World Journal of Gastroenterology : WJG

    doi: 10.3748/wjg.v11.i3.454

    Restriction analysis of pMAL-c2x-napA. Lane 1: 1 kb DNA ladder; Lane 2: pMAL-c2x-napA digested by Eco RI; Lane 3: pMAL-c2x-napA digested by Eco RI and Sal I; Lane 4: pMAL-c2x digested by Eco RI; Lane 5 HP-napA PCR fragment.
    Figure Legend Snippet: Restriction analysis of pMAL-c2x-napA. Lane 1: 1 kb DNA ladder; Lane 2: pMAL-c2x-napA digested by Eco RI; Lane 3: pMAL-c2x-napA digested by Eco RI and Sal I; Lane 4: pMAL-c2x digested by Eco RI; Lane 5 HP-napA PCR fragment.

    Techniques Used: Polymerase Chain Reaction

    10) Product Images from "A series of constitutive expression vectors to accurately measure the rate of DNA transposition and correct for auto-inhibition"

    Article Title: A series of constitutive expression vectors to accurately measure the rate of DNA transposition and correct for auto-inhibition

    Journal: bioRxiv

    doi: 10.1101/423012

    SETMAR transposition activity was lost during the same period as Hsmar1 transposase domestication. A/ Phylogenetic tree of anthropoid primates which represents the apparition of mutations in the Hsmar1 domain of SETMAR. All the mutations present in the human SETMAR were tested by papillation assay to determine their effects on Hsmar1 transposition. B/ Representative colonies of pMAL-C2X expressing wild-type (pRC1721) or mutant Hsmar1 transposases (pRC1877-1899).
    Figure Legend Snippet: SETMAR transposition activity was lost during the same period as Hsmar1 transposase domestication. A/ Phylogenetic tree of anthropoid primates which represents the apparition of mutations in the Hsmar1 domain of SETMAR. All the mutations present in the human SETMAR were tested by papillation assay to determine their effects on Hsmar1 transposition. B/ Representative colonies of pMAL-C2X expressing wild-type (pRC1721) or mutant Hsmar1 transposases (pRC1877-1899).

    Techniques Used: Activity Assay, Expressing, Mutagenesis

    11) Product Images from "Overexpression and purification of U24 from human herpesvirus type-6 in E. coli: unconventional use of oxidizing environments with a maltose binding protein-hexahistine dual tag to enhance membrane protein yield"

    Article Title: Overexpression and purification of U24 from human herpesvirus type-6 in E. coli: unconventional use of oxidizing environments with a maltose binding protein-hexahistine dual tag to enhance membrane protein yield

    Journal: Microbial Cell Factories

    doi: 10.1186/1475-2859-10-51

    Examination of cysteine-free mutant U24 expression . The C21SC37S mutant U24 constructs were expressed: pMAL-p2x-U24 in C41 (DE3) and pMAL-c2x-U24 in Origami 2 cells, at 18°C and 37°C. Removal of disulfide bond potential appeared to have no effect on in vivo stability of expressed MBP-6 × His-U24, which exhibited the same expression characteristics as wild-type; a similar loss in mass of the degraded fusion protein is observed at the higher temperature.
    Figure Legend Snippet: Examination of cysteine-free mutant U24 expression . The C21SC37S mutant U24 constructs were expressed: pMAL-p2x-U24 in C41 (DE3) and pMAL-c2x-U24 in Origami 2 cells, at 18°C and 37°C. Removal of disulfide bond potential appeared to have no effect on in vivo stability of expressed MBP-6 × His-U24, which exhibited the same expression characteristics as wild-type; a similar loss in mass of the degraded fusion protein is observed at the higher temperature.

    Techniques Used: Mutagenesis, Expressing, Construct, In Vivo

    U24 codon-optimized gene, amino acid sequence and graphical representation of expressed protein construct . A) BamHI/HindIII cut sites are indicated and used to clone the PCR-amplified duplex DNA into the corresponding sites of pMAL-p2x and pMAL-c2x vectors, from which the MBP-6 × His-U24 fusion protein is expressed. The U24 gene was designed to be preceded by a hexahistidine tag (6 × His) and LVPRGS thrombin cleavage site (indicated by an arrow). Final thrombin-cleaved and purified U24 protein will include an additional two amino acids (Gly-Ser) at the N-terminus. B) Cartoon representation of expressed protein. The difference in constructs is a signal sequence at the N-terminus of the protein expressed by pMAL-p2x-U24, directing expression to the periplasm. The Factor Xa cleavage site is vector-encoded.
    Figure Legend Snippet: U24 codon-optimized gene, amino acid sequence and graphical representation of expressed protein construct . A) BamHI/HindIII cut sites are indicated and used to clone the PCR-amplified duplex DNA into the corresponding sites of pMAL-p2x and pMAL-c2x vectors, from which the MBP-6 × His-U24 fusion protein is expressed. The U24 gene was designed to be preceded by a hexahistidine tag (6 × His) and LVPRGS thrombin cleavage site (indicated by an arrow). Final thrombin-cleaved and purified U24 protein will include an additional two amino acids (Gly-Ser) at the N-terminus. B) Cartoon representation of expressed protein. The difference in constructs is a signal sequence at the N-terminus of the protein expressed by pMAL-p2x-U24, directing expression to the periplasm. The Factor Xa cleavage site is vector-encoded.

    Techniques Used: Sequencing, Construct, Polymerase Chain Reaction, Amplification, Purification, Expressing, Plasmid Preparation

    12) Product Images from "Alternatively spliced exon 5 of the FERM domain of Protein 4.1R encodes a novel binding site for erythrocyte p55 and is critical for membrane targeting in epithelial cells"

    Article Title: Alternatively spliced exon 5 of the FERM domain of Protein 4.1R encodes a novel binding site for erythrocyte p55 and is critical for membrane targeting in epithelial cells

    Journal: Biochimica et biophysica acta

    doi: 10.1016/j.bbamcr.2008.09.012

    Expression of FERM domain constructs and p55 binding assay. (A) Coomassie blue stained gel showing recombinant MBP fusion proteins used for the binding assays. MBP (lanes 1), MBP-FERMΔExon 5 (lane 2), and MBP-FERM (lane 3). Fusion proteins shown in lanes 1–3 were expressed in the cytoplasm of E. coli using the pMal-c2X expression vector. Fusion proteins were also expressed in the bacterial periplasm using the pMal-p2X vector (data not shown). Results were identical with both preparations. (B) Coomassie blue stained gel showing recombinant MBP (lane 1), MBP-exon 5 (lane 2), and MBP-exon 10 (lane 3) used for the binding studies. These recombinant proteins were expressed in the bacterial cytoplasm. Western blot based detection of His-p55 recovered by the MBP-fusion protein conjugated amylose-beads is shown on the right. A monoclonal antibody raised against human erythrocyte p55 was used for Western blotting.
    Figure Legend Snippet: Expression of FERM domain constructs and p55 binding assay. (A) Coomassie blue stained gel showing recombinant MBP fusion proteins used for the binding assays. MBP (lanes 1), MBP-FERMΔExon 5 (lane 2), and MBP-FERM (lane 3). Fusion proteins shown in lanes 1–3 were expressed in the cytoplasm of E. coli using the pMal-c2X expression vector. Fusion proteins were also expressed in the bacterial periplasm using the pMal-p2X vector (data not shown). Results were identical with both preparations. (B) Coomassie blue stained gel showing recombinant MBP (lane 1), MBP-exon 5 (lane 2), and MBP-exon 10 (lane 3) used for the binding studies. These recombinant proteins were expressed in the bacterial cytoplasm. Western blot based detection of His-p55 recovered by the MBP-fusion protein conjugated amylose-beads is shown on the right. A monoclonal antibody raised against human erythrocyte p55 was used for Western blotting.

    Techniques Used: Expressing, Construct, Binding Assay, Staining, Recombinant, Plasmid Preparation, Western Blot

    13) Product Images from "A novel glycoprotein D-specific monoclonal antibody neutralizes herpes simplex virus"

    Article Title: A novel glycoprotein D-specific monoclonal antibody neutralizes herpes simplex virus

    Journal: Antiviral Research

    doi: 10.1016/j.antiviral.2017.10.013

    Mapping the recognition region of the mAbs against HSV-2 gD protein. (A) Schematic diagram of gD protein and truncation strategy. The structure of full-length gD protein is shown at the top; the truncated gD proteins used for the sequential mapping experiment are shown below. Numbers indicate amino acid positions. The domain (pro-fusion) was defined according to Fusco et al. (2005) . (B) Recognition region mapping of mAbs m27f and 21C11. The truncated gD proteins expressed by pMAL-C2x vector were presented as antigens. m27f and 21C11 were used as the primary antibodies respectively for Western blot analysis.
    Figure Legend Snippet: Mapping the recognition region of the mAbs against HSV-2 gD protein. (A) Schematic diagram of gD protein and truncation strategy. The structure of full-length gD protein is shown at the top; the truncated gD proteins used for the sequential mapping experiment are shown below. Numbers indicate amino acid positions. The domain (pro-fusion) was defined according to Fusco et al. (2005) . (B) Recognition region mapping of mAbs m27f and 21C11. The truncated gD proteins expressed by pMAL-C2x vector were presented as antigens. m27f and 21C11 were used as the primary antibodies respectively for Western blot analysis.

    Techniques Used: Plasmid Preparation, Western Blot

    Detailed epitope mapping of m27f. (A) Schematic diagram of the gD protein and accurate truncation strategy. The structure of the full-length gD protein is shown at the top; the truncated gD proteins used for the sequential mapping experiment are shown below. Numbers indicate amino acid positions. The domain (pro-fusion) was defined according to Fusco et al. (2005) . (B) Epitope mapping of m27f. The truncated gD proteins expressed by pMAL-C2x vector were presented as antigens. MAb m27f was used as the primary antibody for Western blot analysis. (C) Alignment analysis. Residues 292 to 297 are highly conserved in both HSV-1 and HSV-2. Sequence alignment was performed with DNAMAN V6.
    Figure Legend Snippet: Detailed epitope mapping of m27f. (A) Schematic diagram of the gD protein and accurate truncation strategy. The structure of the full-length gD protein is shown at the top; the truncated gD proteins used for the sequential mapping experiment are shown below. Numbers indicate amino acid positions. The domain (pro-fusion) was defined according to Fusco et al. (2005) . (B) Epitope mapping of m27f. The truncated gD proteins expressed by pMAL-C2x vector were presented as antigens. MAb m27f was used as the primary antibody for Western blot analysis. (C) Alignment analysis. Residues 292 to 297 are highly conserved in both HSV-1 and HSV-2. Sequence alignment was performed with DNAMAN V6.

    Techniques Used: Plasmid Preparation, Western Blot, Sequencing

    Related Articles

    Polymerase Chain Reaction:

    Article Title: Chlamydomonas reinhardtii hydin is a central pair protein required for flagellar motility
    Article Snippet: .. The PCR product was digested with HindIII and BamHI and ligated into pMAL-cR1 v. 2 digested with the same enzymes (New England Biolabs, Inc.). .. The construct was transformed into E. coli XL1 blue, and, for expression of the maltose-binding∷hydin fusion protein, into BL21.

    Clone Assay:

    Article Title: The development and application of new crystallization method for tobacco mosaic virus coat protein
    Article Snippet: .. Both plasmid PGEX-6P-1 (Novagen) and CP were digested with BamH I (NEB, 10 units/μL)/Xho I (NEB, 10 units/μL) and cloned into the same sites in PGEX-6P-1 (PGEX-6P-1-WT-GST-TMV-CP32 ). ..

    other:

    Article Title: Probing hyper-negatively supercoiled mini-circles with nucleases and DNA binding proteins
    Article Snippet: Escherichia coli topoisomerase I ( Ec TopoI), T4 polynucleotide kinase (PNK), calf intestinal phosphatase, T4 DNA ligase, DNAse I, BamHI, BglII and HindIII were from New England Biolabs.

    Plasmid Preparation:

    Article Title: The development and application of new crystallization method for tobacco mosaic virus coat protein
    Article Snippet: .. Both plasmid PGEX-6P-1 (Novagen) and CP were digested with BamH I (NEB, 10 units/μL)/Xho I (NEB, 10 units/μL) and cloned into the same sites in PGEX-6P-1 (PGEX-6P-1-WT-GST-TMV-CP32 ). ..

    Article Title: Recognition of DNA Termini by the C-Terminal Region of the Ku80 and the DNA-Dependent Protein Kinase Catalytic Subunit
    Article Snippet: .. Kinase assays using plasmid DNA substrates were performed with pcDNA3.1 digested with either XhoI, BamHI, EcoRV, and KpnI or pCAG-GFP digested with XbaI or EcoRI (New England Biolabs). .. The specific sequences recognized by the restriction enzymes and DNA termini generated are shown in .

    Article Title: Assembly of evolved ligninolytic genes in Saccharomyces cerevisiae
    Article Snippet: .. The ura3 -deficient S. cerevisiae strain BJ5465 ( α ura3–52 trp1 leu2Δ1 his3Δ200 pep4::HIS2 prb1Δ1.6R can1 GAL1 ) was obtained from LGCPromochem, the NucleoSpin Plasmid kit was purchased from Macherey-Nagel, and the restriction enzymes BamHI, NheI, SpeI, SacI, and NotI from New England Biolabs. ..

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    New England Biolabs pmal c2x
    Examination of cysteine-free mutant U24 expression . The C21SC37S mutant U24 constructs were expressed: <t>pMAL-p2x-U24</t> in C41 (DE3) and <t>pMAL-c2x-U24</t> in Origami 2 cells, at 18°C and 37°C. Removal of disulfide bond potential appeared to have no effect on in vivo stability of expressed MBP-6 × His-U24, which exhibited the same expression characteristics as wild-type; a similar loss in mass of the degraded fusion protein is observed at the higher temperature.
    Pmal C2x, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 94/100, based on 218 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pmal c2x/product/New England Biolabs
    Average 94 stars, based on 218 article reviews
    Price from $9.99 to $1999.99
    pmal c2x - by Bioz Stars, 2020-08
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    91
    New England Biolabs expression vector pmal c2x
    Reverse splicing of Dir.S956-1 (EC) in E.coli . ( A ) Diagram of the construct <t>pMAL-Dir.S956-1</t> (EC) used for intron expression in E.coli . An EcoRI–PstI fragment containing the Dir.S956-1 (EC) intron was cloned into the <t>pMAL-c2X</t> expression vector under the control of the IPTG-inducible P tac promoter. Self-splicing of the intron (shown schematically) results in ligated exon sequences and a free intron RNA. ( B ) Schematic presentation of RT–PCR amplification of reverse splicing products. After reverse splicing, integration products were reverse transcribed and amplified by PCR. For 5′ integration junctions, the upstream primer anneals to the 5′ exon and the downstream primer to the intron. For 3′ integration junctions, the upstream primer is targeted to the intron and the downstream primer is complementary to the 3′ exon. ( C ) RT–PCR amplification and sequence analysis of intron– E.coli SSU rRNA junctions. The RNA was from bacterial cells transformed with pMAL-Dir.S956-1 (EC) and induced with IPTG for 2 h. RT–PCR with primers OP621 and OP619 for 5′ integration junctions results in a product of 390 bp, which by sequencing analysis reveals the 5′ end of the intron ligated to U956 of E.coli SSU rRNA. Amplification of the 3′ integration junctions is shown with primers OP85 and OP622. The product of 238 bp represents the 3′ intron integration junction at position S956. Non-specifc annealing of primer OP622 to the pMAL-vector sequence during the RT reaction gave rise to the smaller product of ∼200 bp. The RNA sequences flanking the observed integration junctions (marked with a diamond) at S956 are given with the intron sequence marked in bold capital letters and the rRNA sequence in lower case letters. M, size marker: 1 kb Plus DNA Ladder (Gibco BRL).
    Expression Vector Pmal C2x, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 91/100, based on 65 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 91 stars, based on 65 article reviews
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    Examination of cysteine-free mutant U24 expression . The C21SC37S mutant U24 constructs were expressed: pMAL-p2x-U24 in C41 (DE3) and pMAL-c2x-U24 in Origami 2 cells, at 18°C and 37°C. Removal of disulfide bond potential appeared to have no effect on in vivo stability of expressed MBP-6 × His-U24, which exhibited the same expression characteristics as wild-type; a similar loss in mass of the degraded fusion protein is observed at the higher temperature.

    Journal: Microbial Cell Factories

    Article Title: Overexpression and purification of U24 from human herpesvirus type-6 in E. coli: unconventional use of oxidizing environments with a maltose binding protein-hexahistine dual tag to enhance membrane protein yield

    doi: 10.1186/1475-2859-10-51

    Figure Lengend Snippet: Examination of cysteine-free mutant U24 expression . The C21SC37S mutant U24 constructs were expressed: pMAL-p2x-U24 in C41 (DE3) and pMAL-c2x-U24 in Origami 2 cells, at 18°C and 37°C. Removal of disulfide bond potential appeared to have no effect on in vivo stability of expressed MBP-6 × His-U24, which exhibited the same expression characteristics as wild-type; a similar loss in mass of the degraded fusion protein is observed at the higher temperature.

    Article Snippet: The codon optimized U24 gene was assembled by overlap PCR for insertion into the pMAL-c2x and pMAL-p2x vectors (New England Biolabs).

    Techniques: Mutagenesis, Expressing, Construct, In Vivo

    U24 codon-optimized gene, amino acid sequence and graphical representation of expressed protein construct . A) BamHI/HindIII cut sites are indicated and used to clone the PCR-amplified duplex DNA into the corresponding sites of pMAL-p2x and pMAL-c2x vectors, from which the MBP-6 × His-U24 fusion protein is expressed. The U24 gene was designed to be preceded by a hexahistidine tag (6 × His) and LVPRGS thrombin cleavage site (indicated by an arrow). Final thrombin-cleaved and purified U24 protein will include an additional two amino acids (Gly-Ser) at the N-terminus. B) Cartoon representation of expressed protein. The difference in constructs is a signal sequence at the N-terminus of the protein expressed by pMAL-p2x-U24, directing expression to the periplasm. The Factor Xa cleavage site is vector-encoded.

    Journal: Microbial Cell Factories

    Article Title: Overexpression and purification of U24 from human herpesvirus type-6 in E. coli: unconventional use of oxidizing environments with a maltose binding protein-hexahistine dual tag to enhance membrane protein yield

    doi: 10.1186/1475-2859-10-51

    Figure Lengend Snippet: U24 codon-optimized gene, amino acid sequence and graphical representation of expressed protein construct . A) BamHI/HindIII cut sites are indicated and used to clone the PCR-amplified duplex DNA into the corresponding sites of pMAL-p2x and pMAL-c2x vectors, from which the MBP-6 × His-U24 fusion protein is expressed. The U24 gene was designed to be preceded by a hexahistidine tag (6 × His) and LVPRGS thrombin cleavage site (indicated by an arrow). Final thrombin-cleaved and purified U24 protein will include an additional two amino acids (Gly-Ser) at the N-terminus. B) Cartoon representation of expressed protein. The difference in constructs is a signal sequence at the N-terminus of the protein expressed by pMAL-p2x-U24, directing expression to the periplasm. The Factor Xa cleavage site is vector-encoded.

    Article Snippet: The codon optimized U24 gene was assembled by overlap PCR for insertion into the pMAL-c2x and pMAL-p2x vectors (New England Biolabs).

    Techniques: Sequencing, Construct, Polymerase Chain Reaction, Amplification, Purification, Expressing, Plasmid Preparation

    Expression of FERM domain constructs and p55 binding assay. (A) Coomassie blue stained gel showing recombinant MBP fusion proteins used for the binding assays. MBP (lanes 1), MBP-FERMΔExon 5 (lane 2), and MBP-FERM (lane 3). Fusion proteins shown in lanes 1–3 were expressed in the cytoplasm of E. coli using the pMal-c2X expression vector. Fusion proteins were also expressed in the bacterial periplasm using the pMal-p2X vector (data not shown). Results were identical with both preparations. (B) Coomassie blue stained gel showing recombinant MBP (lane 1), MBP-exon 5 (lane 2), and MBP-exon 10 (lane 3) used for the binding studies. These recombinant proteins were expressed in the bacterial cytoplasm. Western blot based detection of His-p55 recovered by the MBP-fusion protein conjugated amylose-beads is shown on the right. A monoclonal antibody raised against human erythrocyte p55 was used for Western blotting.

    Journal: Biochimica et biophysica acta

    Article Title: Alternatively spliced exon 5 of the FERM domain of Protein 4.1R encodes a novel binding site for erythrocyte p55 and is critical for membrane targeting in epithelial cells

    doi: 10.1016/j.bbamcr.2008.09.012

    Figure Lengend Snippet: Expression of FERM domain constructs and p55 binding assay. (A) Coomassie blue stained gel showing recombinant MBP fusion proteins used for the binding assays. MBP (lanes 1), MBP-FERMΔExon 5 (lane 2), and MBP-FERM (lane 3). Fusion proteins shown in lanes 1–3 were expressed in the cytoplasm of E. coli using the pMal-c2X expression vector. Fusion proteins were also expressed in the bacterial periplasm using the pMal-p2X vector (data not shown). Results were identical with both preparations. (B) Coomassie blue stained gel showing recombinant MBP (lane 1), MBP-exon 5 (lane 2), and MBP-exon 10 (lane 3) used for the binding studies. These recombinant proteins were expressed in the bacterial cytoplasm. Western blot based detection of His-p55 recovered by the MBP-fusion protein conjugated amylose-beads is shown on the right. A monoclonal antibody raised against human erythrocyte p55 was used for Western blotting.

    Article Snippet: The pMal-c2X and pMal-p2X vectors are engineered to facilitate recombinant protein expression in the bacterial cytoplasm and periplasm, respectively (NE BioLabs).

    Techniques: Expressing, Construct, Binding Assay, Staining, Recombinant, Plasmid Preparation, Western Blot

    Reverse splicing of Dir.S956-1 (EC) in E.coli . ( A ) Diagram of the construct pMAL-Dir.S956-1 (EC) used for intron expression in E.coli . An EcoRI–PstI fragment containing the Dir.S956-1 (EC) intron was cloned into the pMAL-c2X expression vector under the control of the IPTG-inducible P tac promoter. Self-splicing of the intron (shown schematically) results in ligated exon sequences and a free intron RNA. ( B ) Schematic presentation of RT–PCR amplification of reverse splicing products. After reverse splicing, integration products were reverse transcribed and amplified by PCR. For 5′ integration junctions, the upstream primer anneals to the 5′ exon and the downstream primer to the intron. For 3′ integration junctions, the upstream primer is targeted to the intron and the downstream primer is complementary to the 3′ exon. ( C ) RT–PCR amplification and sequence analysis of intron– E.coli SSU rRNA junctions. The RNA was from bacterial cells transformed with pMAL-Dir.S956-1 (EC) and induced with IPTG for 2 h. RT–PCR with primers OP621 and OP619 for 5′ integration junctions results in a product of 390 bp, which by sequencing analysis reveals the 5′ end of the intron ligated to U956 of E.coli SSU rRNA. Amplification of the 3′ integration junctions is shown with primers OP85 and OP622. The product of 238 bp represents the 3′ intron integration junction at position S956. Non-specifc annealing of primer OP622 to the pMAL-vector sequence during the RT reaction gave rise to the smaller product of ∼200 bp. The RNA sequences flanking the observed integration junctions (marked with a diamond) at S956 are given with the intron sequence marked in bold capital letters and the rRNA sequence in lower case letters. M, size marker: 1 kb Plus DNA Ladder (Gibco BRL).

    Journal: Nucleic Acids Research

    Article Title: Site-specific reverse splicing of a HEG-containing group I intron in ribosomal RNA

    doi: 10.1093/nar/gki341

    Figure Lengend Snippet: Reverse splicing of Dir.S956-1 (EC) in E.coli . ( A ) Diagram of the construct pMAL-Dir.S956-1 (EC) used for intron expression in E.coli . An EcoRI–PstI fragment containing the Dir.S956-1 (EC) intron was cloned into the pMAL-c2X expression vector under the control of the IPTG-inducible P tac promoter. Self-splicing of the intron (shown schematically) results in ligated exon sequences and a free intron RNA. ( B ) Schematic presentation of RT–PCR amplification of reverse splicing products. After reverse splicing, integration products were reverse transcribed and amplified by PCR. For 5′ integration junctions, the upstream primer anneals to the 5′ exon and the downstream primer to the intron. For 3′ integration junctions, the upstream primer is targeted to the intron and the downstream primer is complementary to the 3′ exon. ( C ) RT–PCR amplification and sequence analysis of intron– E.coli SSU rRNA junctions. The RNA was from bacterial cells transformed with pMAL-Dir.S956-1 (EC) and induced with IPTG for 2 h. RT–PCR with primers OP621 and OP619 for 5′ integration junctions results in a product of 390 bp, which by sequencing analysis reveals the 5′ end of the intron ligated to U956 of E.coli SSU rRNA. Amplification of the 3′ integration junctions is shown with primers OP85 and OP622. The product of 238 bp represents the 3′ intron integration junction at position S956. Non-specifc annealing of primer OP622 to the pMAL-vector sequence during the RT reaction gave rise to the smaller product of ∼200 bp. The RNA sequences flanking the observed integration junctions (marked with a diamond) at S956 are given with the intron sequence marked in bold capital letters and the rRNA sequence in lower case letters. M, size marker: 1 kb Plus DNA Ladder (Gibco BRL).

    Article Snippet: Plasmid construction The plasmid pMAL-Dir.S956-1 (EC), used for expression in E.coli , was prepared by ligating an EcoRI–PstI fragment containing the Didymium twin-ribozyme intron with a mutated IGS (see ) into the expression vector pMAL-c2X (New England Biolabs) leaving the lacZ' translation frame restored after intron splicing.

    Techniques: Construct, Expressing, Clone Assay, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction, Amplification, Polymerase Chain Reaction, Sequencing, Transformation Assay, Marker

    SDS-PAGE electrophoresis on 10 % polyacrylamide gel. 1 E. coli strain TG1 containing pMAL-c2X as control after induction. 2 E. coli extracts expression of pMAL-c2X-GRA7

    Journal: Journal of Parasitic Diseases: Official Organ of the Indian Society for Parasitology

    Article Title: Cloning and expression of Neospora caninum dense-granule 7 in E. coli

    doi: 10.1007/s12639-012-0221-1

    Figure Lengend Snippet: SDS-PAGE electrophoresis on 10 % polyacrylamide gel. 1 E. coli strain TG1 containing pMAL-c2X as control after induction. 2 E. coli extracts expression of pMAL-c2X-GRA7

    Article Snippet: The purified PCR product and pMAL-c2X plasmid (New England Biolabs, USA) were digested with Eco R1 restriction enzymes (Fermentas) at 37 °C for 2.5 h and were extracted from agarose gel using the gel extraction kit.

    Techniques: SDS Page, Electrophoresis, Expressing