Structured Review

GE Healthcare expression vector pgex 2t
Recombinant HMT antigens used for immunizations. a Recombinant human and porcine GST-HMT fusion proteins cloned in the bacterial expression vector <t>pGEX-2T.</t> b 12.5 % Silver-stained polyacrylamide gel of the purified human ( lane 1 ) and porcine ( lane 3 ) GST-HMT fusion proteins used for the immunization of mice and the HMT and GST products resulting from cleavage with thrombin protease ( lanes 2 and 4 ). The sizes of molecular weight markers ( M ) are given on the left in kilodalton. c Sequence alignment and percent sequence identity of the HMT proteins from man, pig, mouse and rat obtained with the NCBI Constrained-based Multiple Alignment Tool ( www.ncbi.nlm.nih.gov/tools/cobalt/cobalt.cgi?link_loc=BlastHomeLink ). Residues identical in all four proteins are shaded black , residues identical in three proteins are shaded gray , and residues that have been shown to interact with histamine and S-adenosylhomocysteine in human HMT [ 7 ] are shaded red (indicated by plus symbol on top) and blue (indicated by hash on top), respectively. Antigenicity plots of human ( d ) and porcine ( e ) HMT produced with the BepiPred Linear Epitope Prediction Tool ( www.tools.immuneepitope.org/bcell ) [ 32 ] show similar predicted B-cell epitopes ( yellow peaks on top of the threshold line with reference to amino acid positions) for both protein sequences
Expression Vector Pgex 2t, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 92/100, based on 27 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Images

1) Product Images from "Monoclonal antibodies for human and porcine histamine N-methyltransferase (HMT) facilitate protein expression and localization studies"

Article Title: Monoclonal antibodies for human and porcine histamine N-methyltransferase (HMT) facilitate protein expression and localization studies

Journal: Inflammation Research

doi: 10.1007/s00011-016-0987-1

Recombinant HMT antigens used for immunizations. a Recombinant human and porcine GST-HMT fusion proteins cloned in the bacterial expression vector pGEX-2T. b 12.5 % Silver-stained polyacrylamide gel of the purified human ( lane 1 ) and porcine ( lane 3 ) GST-HMT fusion proteins used for the immunization of mice and the HMT and GST products resulting from cleavage with thrombin protease ( lanes 2 and 4 ). The sizes of molecular weight markers ( M ) are given on the left in kilodalton. c Sequence alignment and percent sequence identity of the HMT proteins from man, pig, mouse and rat obtained with the NCBI Constrained-based Multiple Alignment Tool ( www.ncbi.nlm.nih.gov/tools/cobalt/cobalt.cgi?link_loc=BlastHomeLink ). Residues identical in all four proteins are shaded black , residues identical in three proteins are shaded gray , and residues that have been shown to interact with histamine and S-adenosylhomocysteine in human HMT [ 7 ] are shaded red (indicated by plus symbol on top) and blue (indicated by hash on top), respectively. Antigenicity plots of human ( d ) and porcine ( e ) HMT produced with the BepiPred Linear Epitope Prediction Tool ( www.tools.immuneepitope.org/bcell ) [ 32 ] show similar predicted B-cell epitopes ( yellow peaks on top of the threshold line with reference to amino acid positions) for both protein sequences
Figure Legend Snippet: Recombinant HMT antigens used for immunizations. a Recombinant human and porcine GST-HMT fusion proteins cloned in the bacterial expression vector pGEX-2T. b 12.5 % Silver-stained polyacrylamide gel of the purified human ( lane 1 ) and porcine ( lane 3 ) GST-HMT fusion proteins used for the immunization of mice and the HMT and GST products resulting from cleavage with thrombin protease ( lanes 2 and 4 ). The sizes of molecular weight markers ( M ) are given on the left in kilodalton. c Sequence alignment and percent sequence identity of the HMT proteins from man, pig, mouse and rat obtained with the NCBI Constrained-based Multiple Alignment Tool ( www.ncbi.nlm.nih.gov/tools/cobalt/cobalt.cgi?link_loc=BlastHomeLink ). Residues identical in all four proteins are shaded black , residues identical in three proteins are shaded gray , and residues that have been shown to interact with histamine and S-adenosylhomocysteine in human HMT [ 7 ] are shaded red (indicated by plus symbol on top) and blue (indicated by hash on top), respectively. Antigenicity plots of human ( d ) and porcine ( e ) HMT produced with the BepiPred Linear Epitope Prediction Tool ( www.tools.immuneepitope.org/bcell ) [ 32 ] show similar predicted B-cell epitopes ( yellow peaks on top of the threshold line with reference to amino acid positions) for both protein sequences

Techniques Used: Recombinant, HMT Assay, Clone Assay, Expressing, Plasmid Preparation, Staining, Purification, Mouse Assay, Molecular Weight, Sequencing, Produced

2) Product Images from "Mapping of the binding sites of human histamine N-methyltransferase (HNMT) monoclonal antibodies"

Article Title: Mapping of the binding sites of human histamine N-methyltransferase (HNMT) monoclonal antibodies

Journal: Inflammation Research

doi: 10.1007/s00011-017-1086-7

HNMT expression constructs. a Recombinant plasmids expressing GST-HNMT fusion proteins obtained by cloning different human HNMT cDNA fragments into the bacterial expression vectors pGEX-2T or pGEX-5X-1/-2, respectively. b 10% silver-stained SDS polyacrylamide gel and immunoblot with an anti-GST antibody of lysates prepared from bacteria harbouring plasmids pGEX-huHMT01-11 indicated on top of each lane . Migration positions of full-length fusion proteins are indicated by arrows and their expected sizes are listed in Table 1 . The sizes of molecular weight markers ( M ) are given on the left in kDa
Figure Legend Snippet: HNMT expression constructs. a Recombinant plasmids expressing GST-HNMT fusion proteins obtained by cloning different human HNMT cDNA fragments into the bacterial expression vectors pGEX-2T or pGEX-5X-1/-2, respectively. b 10% silver-stained SDS polyacrylamide gel and immunoblot with an anti-GST antibody of lysates prepared from bacteria harbouring plasmids pGEX-huHMT01-11 indicated on top of each lane . Migration positions of full-length fusion proteins are indicated by arrows and their expected sizes are listed in Table 1 . The sizes of molecular weight markers ( M ) are given on the left in kDa

Techniques Used: Expressing, Construct, Recombinant, Clone Assay, Staining, Migration, Molecular Weight

3) Product Images from "Expression and purification of 15N- and 13C-isotope labeled 40-residue human Alzheimer's ?-amyloid peptide for NMR-based structural analysis"

Article Title: Expression and purification of 15N- and 13C-isotope labeled 40-residue human Alzheimer's ?-amyloid peptide for NMR-based structural analysis

Journal: Protein expression and purification

doi: 10.1016/j.pep.2011.05.012

Schematic diagram of the expression vector pGEX-2T-Aβ(1–40). Factor Xa recognition site (IEGR) and Aβ(1–40) sequence were inserted into the Bam HI and Eco RI sites of pGEX-2T downstream from GST gene. The vector also had
Figure Legend Snippet: Schematic diagram of the expression vector pGEX-2T-Aβ(1–40). Factor Xa recognition site (IEGR) and Aβ(1–40) sequence were inserted into the Bam HI and Eco RI sites of pGEX-2T downstream from GST gene. The vector also had

Techniques Used: Expressing, Plasmid Preparation, Sequencing

Related Articles

Clone Assay:

Article Title: A surface proton antenna in carbonic anhydrase II supports lactate transport in cancer cells
Article Snippet: .. In brief, the C-termini of MCT1 and MCT4 were cloned into the expression vector pGEX-2T (GE Healthcare Europe GmbH) and transformed into E. coli BL21 cells. .. Protein expression was induced by the addition of 0.8 mM isopropyl-β-D-thiogalactopyranosid (IPTG).

Article Title: Monoclonal antibodies for human and porcine histamine N-methyltransferase (HMT) facilitate protein expression and localization studies
Article Snippet: .. Preparation of recombinant HMT proteins Full-length human and porcine HMT cDNAs [ , , ] were amplified by PCR with specific primers from total human and porcine kidney cDNA, respectively, and cloned in frame into the bacterial expression vector pGEX-2T (GE Healthcare, Vienna, Austria). .. Each recombinant plasmid was transformed into the protease-deficient strain E. coli BL21 to produce glutathione S-transferase (GST) fusion proteins according to manufacturer’s instructions (GE Healthcare, Vienna, Austria).

Article Title: Molecular Interaction Studies of HIV-1 Matrix Protein p17 and Heparin
Article Snippet: .. The coding sequence of HIV-1 matrix protein p17 clade B isolate BH10 (amino acids 1–132) was amplified by PCR with specific primers, allowing the cloning of the p17 sequence into the BamH1 site of the expression vector pGEX-2T (GE Healthcare). p17 N-terminal Lys→Ala mutant, obtained by the substitution of lysines of the N-terminal basic motif with alanine, and the p17 C-terminal K→A mutant, obtained by the same mutations in the C-terminal basic motif, were cloned into the BamH1 site of the pGEX-2T vector using QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA). p17Δ36, obtained by the deletion of the C-terminal sequence 97–132, was prepared as described ( ). .. See A for a schematic representation of the p17 mutants. p17 proteins and the GST motif alone were purified as described ( ).

Article Title: Mapping of the binding sites of human histamine N-methyltransferase (HNMT) monoclonal antibodies
Article Snippet: .. Preparation and expression of recombinant human HNMT protein fragments Full-length human HNMT cDNA [ , ] was amplified by PCR with specific primers from total human kidney cDNA and cloned in frame into the Bam HI site of the bacterial expression vector pGEX-2T (GE Healthcare, Vienna, Austria) to obtain plasmid pGEX-huHMT01 [ ]. .. A series of C-terminal deletions of HNMT was produced by double-digestion of pGEX-huHMT01 with restriction endonucleases Eco RI100 , Xho I322 , Nde I419 , Kpn I491 , Nco I668 , or Bgl II792 (superscripts indicate position of recognition sequence relative to A of translational start codon) that cut once on the cDNA plus Sma I that cuts the vector immediately downstream of the cloning site, creating blunt ends by incubation for 15 min at 37 °C with 1 U Klenow Fragment and 100 µM dNTPs, and religating the respective larger fragments with T4 DNA ligase, resulting in plasmids pGEX-huHMT02-07.

Article Title: Human Milk Contains Novel Glycans That Are Potential Decoy Receptors for Neonatal Rotaviruses *
Article Snippet: .. The gene sequences of VP8* (amino acids 64–225) of neonatal strains N155(G10P[11]) and RV3(G3P[6]) and bovine strain B223(G10P[11]) of RVs were synthesized (Epoch Life Science, Sugarland, TX) and cloned into expression vector pGEX-2T (GE Healthcare). .. The recombinant N-terminal GST-tagged VP8*s were expressed in E. coli BL21 (DE3) (Novagen, Madison, WI) and purified using Thermo Scientific Pierce glutathione agarose.

Article Title: The Shed Ectodomain of Collagen XVII/BP180 Is Targeted by Autoantibodies in Different Blistering Skin Diseases
Article Snippet: .. The fragments were cloned into the 3′ end of the GST gene in the expression vector pGEX-2T (Amersham Pharmacia Biotech), and dideoxynucleotide sequence analysis was performed. .. The constructs were expressed in Escherichia coli DH5α, and the recombinant proteins were purified using the GST-glutathione affinity system according to the manufacturer’s instructions (Amersham Pharmacia Biotech).

Article Title: A surface proton antenna in carbonic anhydrase II supports lactate transport in cancer cells
Article Snippet: .. In brief, the C-termini of MCT1 and MCT4 were cloned into the expression vector pGEX-2T (GE Healthcare Europe GmbH) and transformed into E. coli BL21 cells. .. Protein expression was induced by the addition of 0.8 mM isopropyl-β-D-thiogalactopyranosid (IPTG).

Centrifugation:

Article Title: Monoclonal antibodies for human and porcine histamine N-methyltransferase (HMT) facilitate protein expression and localization studies
Article Snippet: Preparation of recombinant HMT proteins Full-length human and porcine HMT cDNAs [ , , ] were amplified by PCR with specific primers from total human and porcine kidney cDNA, respectively, and cloned in frame into the bacterial expression vector pGEX-2T (GE Healthcare, Vienna, Austria). .. Bacteria were harvested by centrifugation for 5 min at 4000g , 4 °C, washed with cold deionized water, and lysed in lysis buffer (20 mM bis·Tris·HCl, pH 7.0, 5 mM dithiothreitol) containing Complete Protease Inhibitor Cocktail (Roche, Vienna, Germany) using a French Press at 600 psi.

Amplification:

Article Title: Monoclonal antibodies for human and porcine histamine N-methyltransferase (HMT) facilitate protein expression and localization studies
Article Snippet: .. Preparation of recombinant HMT proteins Full-length human and porcine HMT cDNAs [ , , ] were amplified by PCR with specific primers from total human and porcine kidney cDNA, respectively, and cloned in frame into the bacterial expression vector pGEX-2T (GE Healthcare, Vienna, Austria). .. Each recombinant plasmid was transformed into the protease-deficient strain E. coli BL21 to produce glutathione S-transferase (GST) fusion proteins according to manufacturer’s instructions (GE Healthcare, Vienna, Austria).

Article Title: Molecular Interaction Studies of HIV-1 Matrix Protein p17 and Heparin
Article Snippet: .. The coding sequence of HIV-1 matrix protein p17 clade B isolate BH10 (amino acids 1–132) was amplified by PCR with specific primers, allowing the cloning of the p17 sequence into the BamH1 site of the expression vector pGEX-2T (GE Healthcare). p17 N-terminal Lys→Ala mutant, obtained by the substitution of lysines of the N-terminal basic motif with alanine, and the p17 C-terminal K→A mutant, obtained by the same mutations in the C-terminal basic motif, were cloned into the BamH1 site of the pGEX-2T vector using QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA). p17Δ36, obtained by the deletion of the C-terminal sequence 97–132, was prepared as described ( ). .. See A for a schematic representation of the p17 mutants. p17 proteins and the GST motif alone were purified as described ( ).

Article Title: Identification of Small-Molecule Enhancers of Arginine Methylation Catalyzed by Coactivator-Associated Arginine Methyltransferase 1
Article Snippet: .. The construct for human PABP1 encoding residues 437 to 488, PABP1(437–488), was generated by subcloning a PCR amplified fragment from the full length PABP1 gene (a gift from Dr. Bertrand Séraphin at IGBMC) into the expression vector pGEX-2T (GE Healthcare). .. Escherichia coli BL21(DE3) cells transformed with the PABP1(437–488) plasmid were grown in LB at 37 °C.

Article Title: Mapping of the binding sites of human histamine N-methyltransferase (HNMT) monoclonal antibodies
Article Snippet: .. Preparation and expression of recombinant human HNMT protein fragments Full-length human HNMT cDNA [ , ] was amplified by PCR with specific primers from total human kidney cDNA and cloned in frame into the Bam HI site of the bacterial expression vector pGEX-2T (GE Healthcare, Vienna, Austria) to obtain plasmid pGEX-huHMT01 [ ]. .. A series of C-terminal deletions of HNMT was produced by double-digestion of pGEX-huHMT01 with restriction endonucleases Eco RI100 , Xho I322 , Nde I419 , Kpn I491 , Nco I668 , or Bgl II792 (superscripts indicate position of recognition sequence relative to A of translational start codon) that cut once on the cDNA plus Sma I that cuts the vector immediately downstream of the cloning site, creating blunt ends by incubation for 15 min at 37 °C with 1 U Klenow Fragment and 100 µM dNTPs, and religating the respective larger fragments with T4 DNA ligase, resulting in plasmids pGEX-huHMT02-07.

Article Title: The Shed Ectodomain of Collagen XVII/BP180 Is Targeted by Autoantibodies in Different Blistering Skin Diseases
Article Snippet: GST-ColXVII-Ecto3, corresponding to amino acids 1365–1413, was amplified with sense primer 5′-GCG GGATCC GCTGACTTTGCTGGAGATCT-3′; and antisense primer 5′-CGCG GAATTC AGCTGATGCCGGGTGGCCC-3′ (modified from ) and GST-ColXVII-NC16a (NC16a), corresponding to amino acids 490–566, were amplified with the sense primer 5′-GCGC GGATCC GAGGAGGTGAGGAAGCTGAA-3′ and the antisense primer 5′-GCGCG GAATTC AT CCTCGGAGATTTCCATT-3′. .. The fragments were cloned into the 3′ end of the GST gene in the expression vector pGEX-2T (Amersham Pharmacia Biotech), and dideoxynucleotide sequence analysis was performed.

Article Title: Individual Src-family tyrosine kinases direct the degradation or protection of the clock protein Timeless via differential ubiquitylation
Article Snippet: .. The SH3 domains of murine Fyn, Hck, c-Src and c-Yes were amplified by PCR and subcloned into the bacterial expression vector pGEX-2T (GE Healthcare Life Sciences) in-frame with the GST coding region. .. Site-directed mutagenesis (QuikChange II XL System, Stratagene) was used to substitute the codon for the conserved tryptophan residue on the binding surface of each SH3 domain with alanine for use as a negative control.

Synthesized:

Article Title: Human Milk Contains Novel Glycans That Are Potential Decoy Receptors for Neonatal Rotaviruses *
Article Snippet: .. The gene sequences of VP8* (amino acids 64–225) of neonatal strains N155(G10P[11]) and RV3(G3P[6]) and bovine strain B223(G10P[11]) of RVs were synthesized (Epoch Life Science, Sugarland, TX) and cloned into expression vector pGEX-2T (GE Healthcare). .. The recombinant N-terminal GST-tagged VP8*s were expressed in E. coli BL21 (DE3) (Novagen, Madison, WI) and purified using Thermo Scientific Pierce glutathione agarose.

Construct:

Article Title: Identification of Small-Molecule Enhancers of Arginine Methylation Catalyzed by Coactivator-Associated Arginine Methyltransferase 1
Article Snippet: .. The construct for human PABP1 encoding residues 437 to 488, PABP1(437–488), was generated by subcloning a PCR amplified fragment from the full length PABP1 gene (a gift from Dr. Bertrand Séraphin at IGBMC) into the expression vector pGEX-2T (GE Healthcare). .. Escherichia coli BL21(DE3) cells transformed with the PABP1(437–488) plasmid were grown in LB at 37 °C.

Article Title: The Shed Ectodomain of Collagen XVII/BP180 Is Targeted by Autoantibodies in Different Blistering Skin Diseases
Article Snippet: The fragments were cloned into the 3′ end of the GST gene in the expression vector pGEX-2T (Amersham Pharmacia Biotech), and dideoxynucleotide sequence analysis was performed. .. The constructs were expressed in Escherichia coli DH5α, and the recombinant proteins were purified using the GST-glutathione affinity system according to the manufacturer’s instructions (Amersham Pharmacia Biotech).

Article Title: IIp45, an insulin-like growth factor binding protein 2 (IGFBP-2) binding protein, antagonizes IGFBP-2 stimulation of glioma cell invasion
Article Snippet: .. A fusion construct, GST-IIp45, was constructed in the expression vector pGEX-2T (Amersham Pharmacia). .. The fusion protein was then induced in BL21 (DE3) cells by 0.4 mM isopropyl β- d -thiogalactoside for 3hat30°C and purified by using glutathione-Sepharose 4B beads (Amersham Pharmacia) according to the manufacturer's instructions.

Incubation:

Article Title: Mapping of the binding sites of human histamine N-methyltransferase (HNMT) monoclonal antibodies
Article Snippet: Preparation and expression of recombinant human HNMT protein fragments Full-length human HNMT cDNA [ , ] was amplified by PCR with specific primers from total human kidney cDNA and cloned in frame into the Bam HI site of the bacterial expression vector pGEX-2T (GE Healthcare, Vienna, Austria) to obtain plasmid pGEX-huHMT01 [ ]. .. A series of C-terminal deletions of HNMT was produced by double-digestion of pGEX-huHMT01 with restriction endonucleases Eco RI100 , Xho I322 , Nde I419 , Kpn I491 , Nco I668 , or Bgl II792 (superscripts indicate position of recognition sequence relative to A of translational start codon) that cut once on the cDNA plus Sma I that cuts the vector immediately downstream of the cloning site, creating blunt ends by incubation for 15 min at 37 °C with 1 U Klenow Fragment and 100 µM dNTPs, and religating the respective larger fragments with T4 DNA ligase, resulting in plasmids pGEX-huHMT02-07.

Expressing:

Article Title: Expression and purification of 15N- and 13C-isotope labeled 40-residue human Alzheimer's ?-amyloid peptide for NMR-based structural analysis
Article Snippet: .. The expression vector pGEX-2T was purchased from GE Healthcare (Piscataway, NJ). .. Host cell BL21-CodonPlus (DE3) was purchased from Stratagene (La Jolla, CA).

Article Title: A surface proton antenna in carbonic anhydrase II supports lactate transport in cancer cells
Article Snippet: .. In brief, the C-termini of MCT1 and MCT4 were cloned into the expression vector pGEX-2T (GE Healthcare Europe GmbH) and transformed into E. coli BL21 cells. .. Protein expression was induced by the addition of 0.8 mM isopropyl-β-D-thiogalactopyranosid (IPTG).

Article Title: Caenorhabditis elegans Unc-45 Is a Component of Muscle Thick Filaments and Colocalizes with Myosin Heavy Chain B, but Not Myosin Heavy Chain a
Article Snippet: .. Antibody Production and Affinity Purification A portion of the unc-45 cDNA, corresponding to a 58-residue region from amino acid 18 to 76 of the predicted UNC-45 protein, was ligated in frame to the glutathione S -transferase coding region in the expression vector pGEX-2T (Amersham Pharmacia Biotech). .. This glutathione S -transferase::UNC-45 fusion was expressed in E . coli strain BL21(DE3) under standard conditions and purified on glutathione agarose (Amersham Pharmacia Biotech) as described ( ).

Article Title: Monoclonal antibodies for human and porcine histamine N-methyltransferase (HMT) facilitate protein expression and localization studies
Article Snippet: .. Preparation of recombinant HMT proteins Full-length human and porcine HMT cDNAs [ , , ] were amplified by PCR with specific primers from total human and porcine kidney cDNA, respectively, and cloned in frame into the bacterial expression vector pGEX-2T (GE Healthcare, Vienna, Austria). .. Each recombinant plasmid was transformed into the protease-deficient strain E. coli BL21 to produce glutathione S-transferase (GST) fusion proteins according to manufacturer’s instructions (GE Healthcare, Vienna, Austria).

Article Title: Molecular Interaction Studies of HIV-1 Matrix Protein p17 and Heparin
Article Snippet: .. The coding sequence of HIV-1 matrix protein p17 clade B isolate BH10 (amino acids 1–132) was amplified by PCR with specific primers, allowing the cloning of the p17 sequence into the BamH1 site of the expression vector pGEX-2T (GE Healthcare). p17 N-terminal Lys→Ala mutant, obtained by the substitution of lysines of the N-terminal basic motif with alanine, and the p17 C-terminal K→A mutant, obtained by the same mutations in the C-terminal basic motif, were cloned into the BamH1 site of the pGEX-2T vector using QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA). p17Δ36, obtained by the deletion of the C-terminal sequence 97–132, was prepared as described ( ). .. See A for a schematic representation of the p17 mutants. p17 proteins and the GST motif alone were purified as described ( ).

Article Title: Identification of Small-Molecule Enhancers of Arginine Methylation Catalyzed by Coactivator-Associated Arginine Methyltransferase 1
Article Snippet: .. The construct for human PABP1 encoding residues 437 to 488, PABP1(437–488), was generated by subcloning a PCR amplified fragment from the full length PABP1 gene (a gift from Dr. Bertrand Séraphin at IGBMC) into the expression vector pGEX-2T (GE Healthcare). .. Escherichia coli BL21(DE3) cells transformed with the PABP1(437–488) plasmid were grown in LB at 37 °C.

Article Title: Mapping of the binding sites of human histamine N-methyltransferase (HNMT) monoclonal antibodies
Article Snippet: .. Preparation and expression of recombinant human HNMT protein fragments Full-length human HNMT cDNA [ , ] was amplified by PCR with specific primers from total human kidney cDNA and cloned in frame into the Bam HI site of the bacterial expression vector pGEX-2T (GE Healthcare, Vienna, Austria) to obtain plasmid pGEX-huHMT01 [ ]. .. A series of C-terminal deletions of HNMT was produced by double-digestion of pGEX-huHMT01 with restriction endonucleases Eco RI100 , Xho I322 , Nde I419 , Kpn I491 , Nco I668 , or Bgl II792 (superscripts indicate position of recognition sequence relative to A of translational start codon) that cut once on the cDNA plus Sma I that cuts the vector immediately downstream of the cloning site, creating blunt ends by incubation for 15 min at 37 °C with 1 U Klenow Fragment and 100 µM dNTPs, and religating the respective larger fragments with T4 DNA ligase, resulting in plasmids pGEX-huHMT02-07.

Article Title: Human Milk Contains Novel Glycans That Are Potential Decoy Receptors for Neonatal Rotaviruses *
Article Snippet: .. The gene sequences of VP8* (amino acids 64–225) of neonatal strains N155(G10P[11]) and RV3(G3P[6]) and bovine strain B223(G10P[11]) of RVs were synthesized (Epoch Life Science, Sugarland, TX) and cloned into expression vector pGEX-2T (GE Healthcare). .. The recombinant N-terminal GST-tagged VP8*s were expressed in E. coli BL21 (DE3) (Novagen, Madison, WI) and purified using Thermo Scientific Pierce glutathione agarose.

Article Title: The Shed Ectodomain of Collagen XVII/BP180 Is Targeted by Autoantibodies in Different Blistering Skin Diseases
Article Snippet: .. The fragments were cloned into the 3′ end of the GST gene in the expression vector pGEX-2T (Amersham Pharmacia Biotech), and dideoxynucleotide sequence analysis was performed. .. The constructs were expressed in Escherichia coli DH5α, and the recombinant proteins were purified using the GST-glutathione affinity system according to the manufacturer’s instructions (Amersham Pharmacia Biotech).

Article Title: Individual Src-family tyrosine kinases direct the degradation or protection of the clock protein Timeless via differential ubiquitylation
Article Snippet: .. The SH3 domains of murine Fyn, Hck, c-Src and c-Yes were amplified by PCR and subcloned into the bacterial expression vector pGEX-2T (GE Healthcare Life Sciences) in-frame with the GST coding region. .. Site-directed mutagenesis (QuikChange II XL System, Stratagene) was used to substitute the codon for the conserved tryptophan residue on the binding surface of each SH3 domain with alanine for use as a negative control.

Article Title: A surface proton antenna in carbonic anhydrase II supports lactate transport in cancer cells
Article Snippet: .. In brief, the C-termini of MCT1 and MCT4 were cloned into the expression vector pGEX-2T (GE Healthcare Europe GmbH) and transformed into E. coli BL21 cells. .. Protein expression was induced by the addition of 0.8 mM isopropyl-β-D-thiogalactopyranosid (IPTG).

Article Title: IIp45, an insulin-like growth factor binding protein 2 (IGFBP-2) binding protein, antagonizes IGFBP-2 stimulation of glioma cell invasion
Article Snippet: .. A fusion construct, GST-IIp45, was constructed in the expression vector pGEX-2T (Amersham Pharmacia). .. The fusion protein was then induced in BL21 (DE3) cells by 0.4 mM isopropyl β- d -thiogalactoside for 3hat30°C and purified by using glutathione-Sepharose 4B beads (Amersham Pharmacia) according to the manufacturer's instructions.

Modification:

Article Title: The Shed Ectodomain of Collagen XVII/BP180 Is Targeted by Autoantibodies in Different Blistering Skin Diseases
Article Snippet: GST-ColXVII-Ecto3, corresponding to amino acids 1365–1413, was amplified with sense primer 5′-GCG GGATCC GCTGACTTTGCTGGAGATCT-3′; and antisense primer 5′-CGCG GAATTC AGCTGATGCCGGGTGGCCC-3′ (modified from ) and GST-ColXVII-NC16a (NC16a), corresponding to amino acids 490–566, were amplified with the sense primer 5′-GCGC GGATCC GAGGAGGTGAGGAAGCTGAA-3′ and the antisense primer 5′-GCGCG GAATTC AT CCTCGGAGATTTCCATT-3′. .. The fragments were cloned into the 3′ end of the GST gene in the expression vector pGEX-2T (Amersham Pharmacia Biotech), and dideoxynucleotide sequence analysis was performed.

Western Blot:

Article Title: Caenorhabditis elegans Unc-45 Is a Component of Muscle Thick Filaments and Colocalizes with Myosin Heavy Chain B, but Not Myosin Heavy Chain a
Article Snippet: Antibody Production and Affinity Purification A portion of the unc-45 cDNA, corresponding to a 58-residue region from amino acid 18 to 76 of the predicted UNC-45 protein, was ligated in frame to the glutathione S -transferase coding region in the expression vector pGEX-2T (Amersham Pharmacia Biotech). .. The preimmune serum did not detect the putative UNC-45 protein on Western blots and did not show any significant staining in immunofluorescence.

Transformation Assay:

Article Title: A surface proton antenna in carbonic anhydrase II supports lactate transport in cancer cells
Article Snippet: .. In brief, the C-termini of MCT1 and MCT4 were cloned into the expression vector pGEX-2T (GE Healthcare Europe GmbH) and transformed into E. coli BL21 cells. .. Protein expression was induced by the addition of 0.8 mM isopropyl-β-D-thiogalactopyranosid (IPTG).

Article Title: Monoclonal antibodies for human and porcine histamine N-methyltransferase (HMT) facilitate protein expression and localization studies
Article Snippet: Preparation of recombinant HMT proteins Full-length human and porcine HMT cDNAs [ , , ] were amplified by PCR with specific primers from total human and porcine kidney cDNA, respectively, and cloned in frame into the bacterial expression vector pGEX-2T (GE Healthcare, Vienna, Austria). .. Each recombinant plasmid was transformed into the protease-deficient strain E. coli BL21 to produce glutathione S-transferase (GST) fusion proteins according to manufacturer’s instructions (GE Healthcare, Vienna, Austria).

Article Title: Identification of Small-Molecule Enhancers of Arginine Methylation Catalyzed by Coactivator-Associated Arginine Methyltransferase 1
Article Snippet: The construct for human PABP1 encoding residues 437 to 488, PABP1(437–488), was generated by subcloning a PCR amplified fragment from the full length PABP1 gene (a gift from Dr. Bertrand Séraphin at IGBMC) into the expression vector pGEX-2T (GE Healthcare). .. Escherichia coli BL21(DE3) cells transformed with the PABP1(437–488) plasmid were grown in LB at 37 °C.

Article Title: A surface proton antenna in carbonic anhydrase II supports lactate transport in cancer cells
Article Snippet: .. In brief, the C-termini of MCT1 and MCT4 were cloned into the expression vector pGEX-2T (GE Healthcare Europe GmbH) and transformed into E. coli BL21 cells. .. Protein expression was induced by the addition of 0.8 mM isopropyl-β-D-thiogalactopyranosid (IPTG).

Transfection:

Article Title: IIp45, an insulin-like growth factor binding protein 2 (IGFBP-2) binding protein, antagonizes IGFBP-2 stimulation of glioma cell invasion
Article Snippet: A fusion construct, GST-IIp45, was constructed in the expression vector pGEX-2T (Amersham Pharmacia). .. HEK293 cells transfected with IGFBP-2 were lysed with lysis buffer (20 mM Tris·HCl, pH 7.4/150 mM NaCl/1 mM EDTA/0.5% Nonidet P-40/10% glycerol) containing protease inhibitors (Sigma) for 30 min at 4°C.

Protease Inhibitor:

Article Title: A surface proton antenna in carbonic anhydrase II supports lactate transport in cancer cells
Article Snippet: In brief, the C-termini of MCT1 and MCT4 were cloned into the expression vector pGEX-2T (GE Healthcare Europe GmbH) and transformed into E. coli BL21 cells. .. 3 hr after induction, the cells were harvested, resuspended in phosphate-buffered saline (PBS) and lysed in lysis buffer (PBS, supplemented with 2 mM MgCl2 , 1% Triton X-100, and protease inhibitor cocktail tablets from Roche).

Article Title: Monoclonal antibodies for human and porcine histamine N-methyltransferase (HMT) facilitate protein expression and localization studies
Article Snippet: Preparation of recombinant HMT proteins Full-length human and porcine HMT cDNAs [ , , ] were amplified by PCR with specific primers from total human and porcine kidney cDNA, respectively, and cloned in frame into the bacterial expression vector pGEX-2T (GE Healthcare, Vienna, Austria). .. Bacteria were harvested by centrifugation for 5 min at 4000g , 4 °C, washed with cold deionized water, and lysed in lysis buffer (20 mM bis·Tris·HCl, pH 7.0, 5 mM dithiothreitol) containing Complete Protease Inhibitor Cocktail (Roche, Vienna, Germany) using a French Press at 600 psi.

Article Title: A surface proton antenna in carbonic anhydrase II supports lactate transport in cancer cells
Article Snippet: In brief, the C-termini of MCT1 and MCT4 were cloned into the expression vector pGEX-2T (GE Healthcare Europe GmbH) and transformed into E. coli BL21 cells. .. 3 hr after induction, the cells were harvested, resuspended in phosphate-buffered saline (PBS) and lysed in lysis buffer (PBS, supplemented with 2 mM MgCl2 , 1% Triton X-100, and protease inhibitor cocktail tablets from Roche).

Generated:

Article Title: Identification of Small-Molecule Enhancers of Arginine Methylation Catalyzed by Coactivator-Associated Arginine Methyltransferase 1
Article Snippet: .. The construct for human PABP1 encoding residues 437 to 488, PABP1(437–488), was generated by subcloning a PCR amplified fragment from the full length PABP1 gene (a gift from Dr. Bertrand Séraphin at IGBMC) into the expression vector pGEX-2T (GE Healthcare). .. Escherichia coli BL21(DE3) cells transformed with the PABP1(437–488) plasmid were grown in LB at 37 °C.

Article Title: IIp45, an insulin-like growth factor binding protein 2 (IGFBP-2) binding protein, antagonizes IGFBP-2 stimulation of glioma cell invasion
Article Snippet: A rabbit polyclonal antibody raised against an IIp45 epitope (46 NSETPSTPETSSTSL 60) was generated and affinity-purified by Bethyl Laboratories (Montgomery, TX). .. A fusion construct, GST-IIp45, was constructed in the expression vector pGEX-2T (Amersham Pharmacia).

Sequencing:

Article Title: Molecular Interaction Studies of HIV-1 Matrix Protein p17 and Heparin
Article Snippet: .. The coding sequence of HIV-1 matrix protein p17 clade B isolate BH10 (amino acids 1–132) was amplified by PCR with specific primers, allowing the cloning of the p17 sequence into the BamH1 site of the expression vector pGEX-2T (GE Healthcare). p17 N-terminal Lys→Ala mutant, obtained by the substitution of lysines of the N-terminal basic motif with alanine, and the p17 C-terminal K→A mutant, obtained by the same mutations in the C-terminal basic motif, were cloned into the BamH1 site of the pGEX-2T vector using QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA). p17Δ36, obtained by the deletion of the C-terminal sequence 97–132, was prepared as described ( ). .. See A for a schematic representation of the p17 mutants. p17 proteins and the GST motif alone were purified as described ( ).

Article Title: Mapping of the binding sites of human histamine N-methyltransferase (HNMT) monoclonal antibodies
Article Snippet: Preparation and expression of recombinant human HNMT protein fragments Full-length human HNMT cDNA [ , ] was amplified by PCR with specific primers from total human kidney cDNA and cloned in frame into the Bam HI site of the bacterial expression vector pGEX-2T (GE Healthcare, Vienna, Austria) to obtain plasmid pGEX-huHMT01 [ ]. .. A series of C-terminal deletions of HNMT was produced by double-digestion of pGEX-huHMT01 with restriction endonucleases Eco RI100 , Xho I322 , Nde I419 , Kpn I491 , Nco I668 , or Bgl II792 (superscripts indicate position of recognition sequence relative to A of translational start codon) that cut once on the cDNA plus Sma I that cuts the vector immediately downstream of the cloning site, creating blunt ends by incubation for 15 min at 37 °C with 1 U Klenow Fragment and 100 µM dNTPs, and religating the respective larger fragments with T4 DNA ligase, resulting in plasmids pGEX-huHMT02-07.

Article Title: The Shed Ectodomain of Collagen XVII/BP180 Is Targeted by Autoantibodies in Different Blistering Skin Diseases
Article Snippet: .. The fragments were cloned into the 3′ end of the GST gene in the expression vector pGEX-2T (Amersham Pharmacia Biotech), and dideoxynucleotide sequence analysis was performed. .. The constructs were expressed in Escherichia coli DH5α, and the recombinant proteins were purified using the GST-glutathione affinity system according to the manufacturer’s instructions (Amersham Pharmacia Biotech).

Sonication:

Article Title: Identification of Small-Molecule Enhancers of Arginine Methylation Catalyzed by Coactivator-Associated Arginine Methyltransferase 1
Article Snippet: The construct for human PABP1 encoding residues 437 to 488, PABP1(437–488), was generated by subcloning a PCR amplified fragment from the full length PABP1 gene (a gift from Dr. Bertrand Séraphin at IGBMC) into the expression vector pGEX-2T (GE Healthcare). .. Harvested cells were lysed by sonication in 50 mM Tris-HCl pH 8.2, 200 mM NaCl.

Affinity Purification:

Article Title: Caenorhabditis elegans Unc-45 Is a Component of Muscle Thick Filaments and Colocalizes with Myosin Heavy Chain B, but Not Myosin Heavy Chain a
Article Snippet: .. Antibody Production and Affinity Purification A portion of the unc-45 cDNA, corresponding to a 58-residue region from amino acid 18 to 76 of the predicted UNC-45 protein, was ligated in frame to the glutathione S -transferase coding region in the expression vector pGEX-2T (Amersham Pharmacia Biotech). .. This glutathione S -transferase::UNC-45 fusion was expressed in E . coli strain BL21(DE3) under standard conditions and purified on glutathione agarose (Amersham Pharmacia Biotech) as described ( ).

Article Title: IIp45, an insulin-like growth factor binding protein 2 (IGFBP-2) binding protein, antagonizes IGFBP-2 stimulation of glioma cell invasion
Article Snippet: A rabbit polyclonal antibody raised against an IIp45 epitope (46 NSETPSTPETSSTSL 60) was generated and affinity-purified by Bethyl Laboratories (Montgomery, TX). .. A fusion construct, GST-IIp45, was constructed in the expression vector pGEX-2T (Amersham Pharmacia).

Recombinant:

Article Title: Monoclonal antibodies for human and porcine histamine N-methyltransferase (HMT) facilitate protein expression and localization studies
Article Snippet: .. Preparation of recombinant HMT proteins Full-length human and porcine HMT cDNAs [ , , ] were amplified by PCR with specific primers from total human and porcine kidney cDNA, respectively, and cloned in frame into the bacterial expression vector pGEX-2T (GE Healthcare, Vienna, Austria). .. Each recombinant plasmid was transformed into the protease-deficient strain E. coli BL21 to produce glutathione S-transferase (GST) fusion proteins according to manufacturer’s instructions (GE Healthcare, Vienna, Austria).

Article Title: Molecular Interaction Studies of HIV-1 Matrix Protein p17 and Heparin
Article Snippet: Paragraph title: Recombinant Proteins ... The coding sequence of HIV-1 matrix protein p17 clade B isolate BH10 (amino acids 1–132) was amplified by PCR with specific primers, allowing the cloning of the p17 sequence into the BamH1 site of the expression vector pGEX-2T (GE Healthcare). p17 N-terminal Lys→Ala mutant, obtained by the substitution of lysines of the N-terminal basic motif with alanine, and the p17 C-terminal K→A mutant, obtained by the same mutations in the C-terminal basic motif, were cloned into the BamH1 site of the pGEX-2T vector using QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA). p17Δ36, obtained by the deletion of the C-terminal sequence 97–132, was prepared as described ( ).

Article Title: Mapping of the binding sites of human histamine N-methyltransferase (HNMT) monoclonal antibodies
Article Snippet: .. Preparation and expression of recombinant human HNMT protein fragments Full-length human HNMT cDNA [ , ] was amplified by PCR with specific primers from total human kidney cDNA and cloned in frame into the Bam HI site of the bacterial expression vector pGEX-2T (GE Healthcare, Vienna, Austria) to obtain plasmid pGEX-huHMT01 [ ]. .. A series of C-terminal deletions of HNMT was produced by double-digestion of pGEX-huHMT01 with restriction endonucleases Eco RI100 , Xho I322 , Nde I419 , Kpn I491 , Nco I668 , or Bgl II792 (superscripts indicate position of recognition sequence relative to A of translational start codon) that cut once on the cDNA plus Sma I that cuts the vector immediately downstream of the cloning site, creating blunt ends by incubation for 15 min at 37 °C with 1 U Klenow Fragment and 100 µM dNTPs, and religating the respective larger fragments with T4 DNA ligase, resulting in plasmids pGEX-huHMT02-07.

Article Title: Human Milk Contains Novel Glycans That Are Potential Decoy Receptors for Neonatal Rotaviruses *
Article Snippet: The gene sequences of VP8* (amino acids 64–225) of neonatal strains N155(G10P[11]) and RV3(G3P[6]) and bovine strain B223(G10P[11]) of RVs were synthesized (Epoch Life Science, Sugarland, TX) and cloned into expression vector pGEX-2T (GE Healthcare). .. The recombinant N-terminal GST-tagged VP8*s were expressed in E. coli BL21 (DE3) (Novagen, Madison, WI) and purified using Thermo Scientific Pierce glutathione agarose.

Article Title: The Shed Ectodomain of Collagen XVII/BP180 Is Targeted by Autoantibodies in Different Blistering Skin Diseases
Article Snippet: The fragments were cloned into the 3′ end of the GST gene in the expression vector pGEX-2T (Amersham Pharmacia Biotech), and dideoxynucleotide sequence analysis was performed. .. The constructs were expressed in Escherichia coli DH5α, and the recombinant proteins were purified using the GST-glutathione affinity system according to the manufacturer’s instructions (Amersham Pharmacia Biotech).

Immunofluorescence:

Article Title: Caenorhabditis elegans Unc-45 Is a Component of Muscle Thick Filaments and Colocalizes with Myosin Heavy Chain B, but Not Myosin Heavy Chain a
Article Snippet: Antibody Production and Affinity Purification A portion of the unc-45 cDNA, corresponding to a 58-residue region from amino acid 18 to 76 of the predicted UNC-45 protein, was ligated in frame to the glutathione S -transferase coding region in the expression vector pGEX-2T (Amersham Pharmacia Biotech). .. The preimmune serum did not detect the putative UNC-45 protein on Western blots and did not show any significant staining in immunofluorescence.

Article Title: The Shed Ectodomain of Collagen XVII/BP180 Is Targeted by Autoantibodies in Different Blistering Skin Diseases
Article Snippet: The fragments were cloned into the 3′ end of the GST gene in the expression vector pGEX-2T (Amersham Pharmacia Biotech), and dideoxynucleotide sequence analysis was performed. .. Rabbits were immunized with the GST-fusion proteins, using standard immunization protocols (Eurogentec, Ougrée, Belgium), and the specificities of high-titer antisera Ab-ColXVII-NC16a, Ab-ColXVII-Col15-2, and Ab-ColXVII-Ecto3 were tested with immunoblotting and immunofluorescence methods.

Mutagenesis:

Article Title: Molecular Interaction Studies of HIV-1 Matrix Protein p17 and Heparin
Article Snippet: .. The coding sequence of HIV-1 matrix protein p17 clade B isolate BH10 (amino acids 1–132) was amplified by PCR with specific primers, allowing the cloning of the p17 sequence into the BamH1 site of the expression vector pGEX-2T (GE Healthcare). p17 N-terminal Lys→Ala mutant, obtained by the substitution of lysines of the N-terminal basic motif with alanine, and the p17 C-terminal K→A mutant, obtained by the same mutations in the C-terminal basic motif, were cloned into the BamH1 site of the pGEX-2T vector using QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA). p17Δ36, obtained by the deletion of the C-terminal sequence 97–132, was prepared as described ( ). .. See A for a schematic representation of the p17 mutants. p17 proteins and the GST motif alone were purified as described ( ).

Article Title: Individual Src-family tyrosine kinases direct the degradation or protection of the clock protein Timeless via differential ubiquitylation
Article Snippet: The SH3 domains of murine Fyn, Hck, c-Src and c-Yes were amplified by PCR and subcloned into the bacterial expression vector pGEX-2T (GE Healthcare Life Sciences) in-frame with the GST coding region. .. Site-directed mutagenesis (QuikChange II XL System, Stratagene) was used to substitute the codon for the conserved tryptophan residue on the binding surface of each SH3 domain with alanine for use as a negative control.

Subcloning:

Article Title: Identification of Small-Molecule Enhancers of Arginine Methylation Catalyzed by Coactivator-Associated Arginine Methyltransferase 1
Article Snippet: .. The construct for human PABP1 encoding residues 437 to 488, PABP1(437–488), was generated by subcloning a PCR amplified fragment from the full length PABP1 gene (a gift from Dr. Bertrand Séraphin at IGBMC) into the expression vector pGEX-2T (GE Healthcare). .. Escherichia coli BL21(DE3) cells transformed with the PABP1(437–488) plasmid were grown in LB at 37 °C.

Size-exclusion Chromatography:

Article Title: Human Milk Contains Novel Glycans That Are Potential Decoy Receptors for Neonatal Rotaviruses *
Article Snippet: The gene sequences of VP8* (amino acids 64–225) of neonatal strains N155(G10P[11]) and RV3(G3P[6]) and bovine strain B223(G10P[11]) of RVs were synthesized (Epoch Life Science, Sugarland, TX) and cloned into expression vector pGEX-2T (GE Healthcare). .. The GST-VP8*s were concentrated using Spin-X UF 20 centrifugal concentrators (Corning, Corning, NY) and further purified via size exclusion chromatography on a High Load 16/60 Superdex 200 (GE Healthcare) column with 10 m m Tris, pH 7.4, 100 m m NaCl at 4 °C.

Labeling:

Article Title: IIp45, an insulin-like growth factor binding protein 2 (IGFBP-2) binding protein, antagonizes IGFBP-2 stimulation of glioma cell invasion
Article Snippet: Labeling was carried out with the RediPrime II random prime labeling system (Amersham Pharmacia). .. A fusion construct, GST-IIp45, was constructed in the expression vector pGEX-2T (Amersham Pharmacia).

Purification:

Article Title: Caenorhabditis elegans Unc-45 Is a Component of Muscle Thick Filaments and Colocalizes with Myosin Heavy Chain B, but Not Myosin Heavy Chain a
Article Snippet: Antibody Production and Affinity Purification A portion of the unc-45 cDNA, corresponding to a 58-residue region from amino acid 18 to 76 of the predicted UNC-45 protein, was ligated in frame to the glutathione S -transferase coding region in the expression vector pGEX-2T (Amersham Pharmacia Biotech). .. This glutathione S -transferase::UNC-45 fusion was expressed in E . coli strain BL21(DE3) under standard conditions and purified on glutathione agarose (Amersham Pharmacia Biotech) as described ( ).

Article Title: Molecular Interaction Studies of HIV-1 Matrix Protein p17 and Heparin
Article Snippet: The coding sequence of HIV-1 matrix protein p17 clade B isolate BH10 (amino acids 1–132) was amplified by PCR with specific primers, allowing the cloning of the p17 sequence into the BamH1 site of the expression vector pGEX-2T (GE Healthcare). p17 N-terminal Lys→Ala mutant, obtained by the substitution of lysines of the N-terminal basic motif with alanine, and the p17 C-terminal K→A mutant, obtained by the same mutations in the C-terminal basic motif, were cloned into the BamH1 site of the pGEX-2T vector using QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA). p17Δ36, obtained by the deletion of the C-terminal sequence 97–132, was prepared as described ( ). .. See A for a schematic representation of the p17 mutants. p17 proteins and the GST motif alone were purified as described ( ).

Article Title: Identification of Small-Molecule Enhancers of Arginine Methylation Catalyzed by Coactivator-Associated Arginine Methyltransferase 1
Article Snippet: Paragraph title: GST-PABP1(437–488) expression and purification ... The construct for human PABP1 encoding residues 437 to 488, PABP1(437–488), was generated by subcloning a PCR amplified fragment from the full length PABP1 gene (a gift from Dr. Bertrand Séraphin at IGBMC) into the expression vector pGEX-2T (GE Healthcare).

Article Title: Human Milk Contains Novel Glycans That Are Potential Decoy Receptors for Neonatal Rotaviruses *
Article Snippet: The gene sequences of VP8* (amino acids 64–225) of neonatal strains N155(G10P[11]) and RV3(G3P[6]) and bovine strain B223(G10P[11]) of RVs were synthesized (Epoch Life Science, Sugarland, TX) and cloned into expression vector pGEX-2T (GE Healthcare). .. The recombinant N-terminal GST-tagged VP8*s were expressed in E. coli BL21 (DE3) (Novagen, Madison, WI) and purified using Thermo Scientific Pierce glutathione agarose.

Article Title: The Shed Ectodomain of Collagen XVII/BP180 Is Targeted by Autoantibodies in Different Blistering Skin Diseases
Article Snippet: The fragments were cloned into the 3′ end of the GST gene in the expression vector pGEX-2T (Amersham Pharmacia Biotech), and dideoxynucleotide sequence analysis was performed. .. The constructs were expressed in Escherichia coli DH5α, and the recombinant proteins were purified using the GST-glutathione affinity system according to the manufacturer’s instructions (Amersham Pharmacia Biotech).

Article Title: IIp45, an insulin-like growth factor binding protein 2 (IGFBP-2) binding protein, antagonizes IGFBP-2 stimulation of glioma cell invasion
Article Snippet: A fusion construct, GST-IIp45, was constructed in the expression vector pGEX-2T (Amersham Pharmacia). .. The fusion protein was then induced in BL21 (DE3) cells by 0.4 mM isopropyl β- d -thiogalactoside for 3hat30°C and purified by using glutathione-Sepharose 4B beads (Amersham Pharmacia) according to the manufacturer's instructions.

Polymerase Chain Reaction:

Article Title: Monoclonal antibodies for human and porcine histamine N-methyltransferase (HMT) facilitate protein expression and localization studies
Article Snippet: .. Preparation of recombinant HMT proteins Full-length human and porcine HMT cDNAs [ , , ] were amplified by PCR with specific primers from total human and porcine kidney cDNA, respectively, and cloned in frame into the bacterial expression vector pGEX-2T (GE Healthcare, Vienna, Austria). .. Each recombinant plasmid was transformed into the protease-deficient strain E. coli BL21 to produce glutathione S-transferase (GST) fusion proteins according to manufacturer’s instructions (GE Healthcare, Vienna, Austria).

Article Title: Molecular Interaction Studies of HIV-1 Matrix Protein p17 and Heparin
Article Snippet: .. The coding sequence of HIV-1 matrix protein p17 clade B isolate BH10 (amino acids 1–132) was amplified by PCR with specific primers, allowing the cloning of the p17 sequence into the BamH1 site of the expression vector pGEX-2T (GE Healthcare). p17 N-terminal Lys→Ala mutant, obtained by the substitution of lysines of the N-terminal basic motif with alanine, and the p17 C-terminal K→A mutant, obtained by the same mutations in the C-terminal basic motif, were cloned into the BamH1 site of the pGEX-2T vector using QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA). p17Δ36, obtained by the deletion of the C-terminal sequence 97–132, was prepared as described ( ). .. See A for a schematic representation of the p17 mutants. p17 proteins and the GST motif alone were purified as described ( ).

Article Title: Identification of Small-Molecule Enhancers of Arginine Methylation Catalyzed by Coactivator-Associated Arginine Methyltransferase 1
Article Snippet: .. The construct for human PABP1 encoding residues 437 to 488, PABP1(437–488), was generated by subcloning a PCR amplified fragment from the full length PABP1 gene (a gift from Dr. Bertrand Séraphin at IGBMC) into the expression vector pGEX-2T (GE Healthcare). .. Escherichia coli BL21(DE3) cells transformed with the PABP1(437–488) plasmid were grown in LB at 37 °C.

Article Title: Mapping of the binding sites of human histamine N-methyltransferase (HNMT) monoclonal antibodies
Article Snippet: .. Preparation and expression of recombinant human HNMT protein fragments Full-length human HNMT cDNA [ , ] was amplified by PCR with specific primers from total human kidney cDNA and cloned in frame into the Bam HI site of the bacterial expression vector pGEX-2T (GE Healthcare, Vienna, Austria) to obtain plasmid pGEX-huHMT01 [ ]. .. A series of C-terminal deletions of HNMT was produced by double-digestion of pGEX-huHMT01 with restriction endonucleases Eco RI100 , Xho I322 , Nde I419 , Kpn I491 , Nco I668 , or Bgl II792 (superscripts indicate position of recognition sequence relative to A of translational start codon) that cut once on the cDNA plus Sma I that cuts the vector immediately downstream of the cloning site, creating blunt ends by incubation for 15 min at 37 °C with 1 U Klenow Fragment and 100 µM dNTPs, and religating the respective larger fragments with T4 DNA ligase, resulting in plasmids pGEX-huHMT02-07.

Article Title: The Shed Ectodomain of Collagen XVII/BP180 Is Targeted by Autoantibodies in Different Blistering Skin Diseases
Article Snippet: For the production of bacterial fusion proteins with the glutathione S -transferase (GST) Gene Fusion System (Amersham Pharmacia Biotech, Uppsala, Sweden), three different collagen XVII cDNA fragments were amplified from human keratinocyte mRNA by Titan reverse transcriptase PCR (Boehringer Mannheim), using sense primers with Bam HI and antisense primers with Eco RI restriction sites (underlined). .. The fragments were cloned into the 3′ end of the GST gene in the expression vector pGEX-2T (Amersham Pharmacia Biotech), and dideoxynucleotide sequence analysis was performed.

Article Title: Individual Src-family tyrosine kinases direct the degradation or protection of the clock protein Timeless via differential ubiquitylation
Article Snippet: .. The SH3 domains of murine Fyn, Hck, c-Src and c-Yes were amplified by PCR and subcloned into the bacterial expression vector pGEX-2T (GE Healthcare Life Sciences) in-frame with the GST coding region. .. Site-directed mutagenesis (QuikChange II XL System, Stratagene) was used to substitute the codon for the conserved tryptophan residue on the binding surface of each SH3 domain with alanine for use as a negative control.

Lysis:

Article Title: A surface proton antenna in carbonic anhydrase II supports lactate transport in cancer cells
Article Snippet: In brief, the C-termini of MCT1 and MCT4 were cloned into the expression vector pGEX-2T (GE Healthcare Europe GmbH) and transformed into E. coli BL21 cells. .. 3 hr after induction, the cells were harvested, resuspended in phosphate-buffered saline (PBS) and lysed in lysis buffer (PBS, supplemented with 2 mM MgCl2 , 1% Triton X-100, and protease inhibitor cocktail tablets from Roche).

Article Title: Monoclonal antibodies for human and porcine histamine N-methyltransferase (HMT) facilitate protein expression and localization studies
Article Snippet: Preparation of recombinant HMT proteins Full-length human and porcine HMT cDNAs [ , , ] were amplified by PCR with specific primers from total human and porcine kidney cDNA, respectively, and cloned in frame into the bacterial expression vector pGEX-2T (GE Healthcare, Vienna, Austria). .. Bacteria were harvested by centrifugation for 5 min at 4000g , 4 °C, washed with cold deionized water, and lysed in lysis buffer (20 mM bis·Tris·HCl, pH 7.0, 5 mM dithiothreitol) containing Complete Protease Inhibitor Cocktail (Roche, Vienna, Germany) using a French Press at 600 psi.

Article Title: A surface proton antenna in carbonic anhydrase II supports lactate transport in cancer cells
Article Snippet: In brief, the C-termini of MCT1 and MCT4 were cloned into the expression vector pGEX-2T (GE Healthcare Europe GmbH) and transformed into E. coli BL21 cells. .. 3 hr after induction, the cells were harvested, resuspended in phosphate-buffered saline (PBS) and lysed in lysis buffer (PBS, supplemented with 2 mM MgCl2 , 1% Triton X-100, and protease inhibitor cocktail tablets from Roche).

Article Title: IIp45, an insulin-like growth factor binding protein 2 (IGFBP-2) binding protein, antagonizes IGFBP-2 stimulation of glioma cell invasion
Article Snippet: A fusion construct, GST-IIp45, was constructed in the expression vector pGEX-2T (Amersham Pharmacia). .. HEK293 cells transfected with IGFBP-2 were lysed with lysis buffer (20 mM Tris·HCl, pH 7.4/150 mM NaCl/1 mM EDTA/0.5% Nonidet P-40/10% glycerol) containing protease inhibitors (Sigma) for 30 min at 4°C.

Plasmid Preparation:

Article Title: Expression and purification of 15N- and 13C-isotope labeled 40-residue human Alzheimer's ?-amyloid peptide for NMR-based structural analysis
Article Snippet: .. The expression vector pGEX-2T was purchased from GE Healthcare (Piscataway, NJ). .. Host cell BL21-CodonPlus (DE3) was purchased from Stratagene (La Jolla, CA).

Article Title: A surface proton antenna in carbonic anhydrase II supports lactate transport in cancer cells
Article Snippet: .. In brief, the C-termini of MCT1 and MCT4 were cloned into the expression vector pGEX-2T (GE Healthcare Europe GmbH) and transformed into E. coli BL21 cells. .. Protein expression was induced by the addition of 0.8 mM isopropyl-β-D-thiogalactopyranosid (IPTG).

Article Title: Caenorhabditis elegans Unc-45 Is a Component of Muscle Thick Filaments and Colocalizes with Myosin Heavy Chain B, but Not Myosin Heavy Chain a
Article Snippet: .. Antibody Production and Affinity Purification A portion of the unc-45 cDNA, corresponding to a 58-residue region from amino acid 18 to 76 of the predicted UNC-45 protein, was ligated in frame to the glutathione S -transferase coding region in the expression vector pGEX-2T (Amersham Pharmacia Biotech). .. This glutathione S -transferase::UNC-45 fusion was expressed in E . coli strain BL21(DE3) under standard conditions and purified on glutathione agarose (Amersham Pharmacia Biotech) as described ( ).

Article Title: Monoclonal antibodies for human and porcine histamine N-methyltransferase (HMT) facilitate protein expression and localization studies
Article Snippet: .. Preparation of recombinant HMT proteins Full-length human and porcine HMT cDNAs [ , , ] were amplified by PCR with specific primers from total human and porcine kidney cDNA, respectively, and cloned in frame into the bacterial expression vector pGEX-2T (GE Healthcare, Vienna, Austria). .. Each recombinant plasmid was transformed into the protease-deficient strain E. coli BL21 to produce glutathione S-transferase (GST) fusion proteins according to manufacturer’s instructions (GE Healthcare, Vienna, Austria).

Article Title: Molecular Interaction Studies of HIV-1 Matrix Protein p17 and Heparin
Article Snippet: .. The coding sequence of HIV-1 matrix protein p17 clade B isolate BH10 (amino acids 1–132) was amplified by PCR with specific primers, allowing the cloning of the p17 sequence into the BamH1 site of the expression vector pGEX-2T (GE Healthcare). p17 N-terminal Lys→Ala mutant, obtained by the substitution of lysines of the N-terminal basic motif with alanine, and the p17 C-terminal K→A mutant, obtained by the same mutations in the C-terminal basic motif, were cloned into the BamH1 site of the pGEX-2T vector using QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA). p17Δ36, obtained by the deletion of the C-terminal sequence 97–132, was prepared as described ( ). .. See A for a schematic representation of the p17 mutants. p17 proteins and the GST motif alone were purified as described ( ).

Article Title: Identification of Small-Molecule Enhancers of Arginine Methylation Catalyzed by Coactivator-Associated Arginine Methyltransferase 1
Article Snippet: .. The construct for human PABP1 encoding residues 437 to 488, PABP1(437–488), was generated by subcloning a PCR amplified fragment from the full length PABP1 gene (a gift from Dr. Bertrand Séraphin at IGBMC) into the expression vector pGEX-2T (GE Healthcare). .. Escherichia coli BL21(DE3) cells transformed with the PABP1(437–488) plasmid were grown in LB at 37 °C.

Article Title: Mapping of the binding sites of human histamine N-methyltransferase (HNMT) monoclonal antibodies
Article Snippet: .. Preparation and expression of recombinant human HNMT protein fragments Full-length human HNMT cDNA [ , ] was amplified by PCR with specific primers from total human kidney cDNA and cloned in frame into the Bam HI site of the bacterial expression vector pGEX-2T (GE Healthcare, Vienna, Austria) to obtain plasmid pGEX-huHMT01 [ ]. .. A series of C-terminal deletions of HNMT was produced by double-digestion of pGEX-huHMT01 with restriction endonucleases Eco RI100 , Xho I322 , Nde I419 , Kpn I491 , Nco I668 , or Bgl II792 (superscripts indicate position of recognition sequence relative to A of translational start codon) that cut once on the cDNA plus Sma I that cuts the vector immediately downstream of the cloning site, creating blunt ends by incubation for 15 min at 37 °C with 1 U Klenow Fragment and 100 µM dNTPs, and religating the respective larger fragments with T4 DNA ligase, resulting in plasmids pGEX-huHMT02-07.

Article Title: Human Milk Contains Novel Glycans That Are Potential Decoy Receptors for Neonatal Rotaviruses *
Article Snippet: .. The gene sequences of VP8* (amino acids 64–225) of neonatal strains N155(G10P[11]) and RV3(G3P[6]) and bovine strain B223(G10P[11]) of RVs were synthesized (Epoch Life Science, Sugarland, TX) and cloned into expression vector pGEX-2T (GE Healthcare). .. The recombinant N-terminal GST-tagged VP8*s were expressed in E. coli BL21 (DE3) (Novagen, Madison, WI) and purified using Thermo Scientific Pierce glutathione agarose.

Article Title: The Shed Ectodomain of Collagen XVII/BP180 Is Targeted by Autoantibodies in Different Blistering Skin Diseases
Article Snippet: .. The fragments were cloned into the 3′ end of the GST gene in the expression vector pGEX-2T (Amersham Pharmacia Biotech), and dideoxynucleotide sequence analysis was performed. .. The constructs were expressed in Escherichia coli DH5α, and the recombinant proteins were purified using the GST-glutathione affinity system according to the manufacturer’s instructions (Amersham Pharmacia Biotech).

Article Title: Individual Src-family tyrosine kinases direct the degradation or protection of the clock protein Timeless via differential ubiquitylation
Article Snippet: .. The SH3 domains of murine Fyn, Hck, c-Src and c-Yes were amplified by PCR and subcloned into the bacterial expression vector pGEX-2T (GE Healthcare Life Sciences) in-frame with the GST coding region. .. Site-directed mutagenesis (QuikChange II XL System, Stratagene) was used to substitute the codon for the conserved tryptophan residue on the binding surface of each SH3 domain with alanine for use as a negative control.

Article Title: A surface proton antenna in carbonic anhydrase II supports lactate transport in cancer cells
Article Snippet: .. In brief, the C-termini of MCT1 and MCT4 were cloned into the expression vector pGEX-2T (GE Healthcare Europe GmbH) and transformed into E. coli BL21 cells. .. Protein expression was induced by the addition of 0.8 mM isopropyl-β-D-thiogalactopyranosid (IPTG).

Article Title: IIp45, an insulin-like growth factor binding protein 2 (IGFBP-2) binding protein, antagonizes IGFBP-2 stimulation of glioma cell invasion
Article Snippet: .. A fusion construct, GST-IIp45, was constructed in the expression vector pGEX-2T (Amersham Pharmacia). .. The fusion protein was then induced in BL21 (DE3) cells by 0.4 mM isopropyl β- d -thiogalactoside for 3hat30°C and purified by using glutathione-Sepharose 4B beads (Amersham Pharmacia) according to the manufacturer's instructions.

Software:

Article Title: Human Milk Contains Novel Glycans That Are Potential Decoy Receptors for Neonatal Rotaviruses *
Article Snippet: The gene sequences of VP8* (amino acids 64–225) of neonatal strains N155(G10P[11]) and RV3(G3P[6]) and bovine strain B223(G10P[11]) of RVs were synthesized (Epoch Life Science, Sugarland, TX) and cloned into expression vector pGEX-2T (GE Healthcare). .. The concentration of purified GST-VP8* was determined by measuring absorbance at 280 nm and using absorption coefficients of 76,270 M−1 cm−1 (N155), 73,000 M−1 cm−1 (RV3), and 73,830 M−1 cm−1 (B223), calculated using Vector NTI 11 software (Invitrogen).

Negative Control:

Article Title: Individual Src-family tyrosine kinases direct the degradation or protection of the clock protein Timeless via differential ubiquitylation
Article Snippet: The SH3 domains of murine Fyn, Hck, c-Src and c-Yes were amplified by PCR and subcloned into the bacterial expression vector pGEX-2T (GE Healthcare Life Sciences) in-frame with the GST coding region. .. Site-directed mutagenesis (QuikChange II XL System, Stratagene) was used to substitute the codon for the conserved tryptophan residue on the binding surface of each SH3 domain with alanine for use as a negative control.

Binding Assay:

Article Title: Individual Src-family tyrosine kinases direct the degradation or protection of the clock protein Timeless via differential ubiquitylation
Article Snippet: The SH3 domains of murine Fyn, Hck, c-Src and c-Yes were amplified by PCR and subcloned into the bacterial expression vector pGEX-2T (GE Healthcare Life Sciences) in-frame with the GST coding region. .. Site-directed mutagenesis (QuikChange II XL System, Stratagene) was used to substitute the codon for the conserved tryptophan residue on the binding surface of each SH3 domain with alanine for use as a negative control.

Article Title: IIp45, an insulin-like growth factor binding protein 2 (IGFBP-2) binding protein, antagonizes IGFBP-2 stimulation of glioma cell invasion
Article Snippet: A fusion construct, GST-IIp45, was constructed in the expression vector pGEX-2T (Amersham Pharmacia). .. Binding assays were carried out as described ( ) with some minor modifications.

Produced:

Article Title: Mapping of the binding sites of human histamine N-methyltransferase (HNMT) monoclonal antibodies
Article Snippet: Preparation and expression of recombinant human HNMT protein fragments Full-length human HNMT cDNA [ , ] was amplified by PCR with specific primers from total human kidney cDNA and cloned in frame into the Bam HI site of the bacterial expression vector pGEX-2T (GE Healthcare, Vienna, Austria) to obtain plasmid pGEX-huHMT01 [ ]. .. A series of C-terminal deletions of HNMT was produced by double-digestion of pGEX-huHMT01 with restriction endonucleases Eco RI100 , Xho I322 , Nde I419 , Kpn I491 , Nco I668 , or Bgl II792 (superscripts indicate position of recognition sequence relative to A of translational start codon) that cut once on the cDNA plus Sma I that cuts the vector immediately downstream of the cloning site, creating blunt ends by incubation for 15 min at 37 °C with 1 U Klenow Fragment and 100 µM dNTPs, and religating the respective larger fragments with T4 DNA ligase, resulting in plasmids pGEX-huHMT02-07.

Concentration Assay:

Article Title: Human Milk Contains Novel Glycans That Are Potential Decoy Receptors for Neonatal Rotaviruses *
Article Snippet: The gene sequences of VP8* (amino acids 64–225) of neonatal strains N155(G10P[11]) and RV3(G3P[6]) and bovine strain B223(G10P[11]) of RVs were synthesized (Epoch Life Science, Sugarland, TX) and cloned into expression vector pGEX-2T (GE Healthcare). .. The concentration of purified GST-VP8* was determined by measuring absorbance at 280 nm and using absorption coefficients of 76,270 M−1 cm−1 (N155), 73,000 M−1 cm−1 (RV3), and 73,830 M−1 cm−1 (B223), calculated using Vector NTI 11 software (Invitrogen).

Staining:

Article Title: Caenorhabditis elegans Unc-45 Is a Component of Muscle Thick Filaments and Colocalizes with Myosin Heavy Chain B, but Not Myosin Heavy Chain a
Article Snippet: Antibody Production and Affinity Purification A portion of the unc-45 cDNA, corresponding to a 58-residue region from amino acid 18 to 76 of the predicted UNC-45 protein, was ligated in frame to the glutathione S -transferase coding region in the expression vector pGEX-2T (Amersham Pharmacia Biotech). .. The preimmune serum did not detect the putative UNC-45 protein on Western blots and did not show any significant staining in immunofluorescence.

HMT Assay:

Article Title: Monoclonal antibodies for human and porcine histamine N-methyltransferase (HMT) facilitate protein expression and localization studies
Article Snippet: .. Preparation of recombinant HMT proteins Full-length human and porcine HMT cDNAs [ , , ] were amplified by PCR with specific primers from total human and porcine kidney cDNA, respectively, and cloned in frame into the bacterial expression vector pGEX-2T (GE Healthcare, Vienna, Austria). .. Each recombinant plasmid was transformed into the protease-deficient strain E. coli BL21 to produce glutathione S-transferase (GST) fusion proteins according to manufacturer’s instructions (GE Healthcare, Vienna, Austria).

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    GE Healthcare expression vector pgex 2t
    Recombinant HMT antigens used for immunizations. a Recombinant human and porcine GST-HMT fusion proteins cloned in the bacterial expression vector <t>pGEX-2T.</t> b 12.5 % Silver-stained polyacrylamide gel of the purified human ( lane 1 ) and porcine ( lane 3 ) GST-HMT fusion proteins used for the immunization of mice and the HMT and GST products resulting from cleavage with thrombin protease ( lanes 2 and 4 ). The sizes of molecular weight markers ( M ) are given on the left in kilodalton. c Sequence alignment and percent sequence identity of the HMT proteins from man, pig, mouse and rat obtained with the NCBI Constrained-based Multiple Alignment Tool ( www.ncbi.nlm.nih.gov/tools/cobalt/cobalt.cgi?link_loc=BlastHomeLink ). Residues identical in all four proteins are shaded black , residues identical in three proteins are shaded gray , and residues that have been shown to interact with histamine and S-adenosylhomocysteine in human HMT [ 7 ] are shaded red (indicated by plus symbol on top) and blue (indicated by hash on top), respectively. Antigenicity plots of human ( d ) and porcine ( e ) HMT produced with the BepiPred Linear Epitope Prediction Tool ( www.tools.immuneepitope.org/bcell ) [ 32 ] show similar predicted B-cell epitopes ( yellow peaks on top of the threshold line with reference to amino acid positions) for both protein sequences
    Expression Vector Pgex 2t, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 92/100, based on 27 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/expression vector pgex 2t/product/GE Healthcare
    Average 92 stars, based on 27 article reviews
    Price from $9.99 to $1999.99
    expression vector pgex 2t - by Bioz Stars, 2020-04
    92/100 stars
      Buy from Supplier

    87
    GE Healthcare prokaryotic expression vector pgex 2t
    Recombinant HMT antigens used for immunizations. a Recombinant human and porcine GST-HMT fusion proteins cloned in the bacterial expression vector <t>pGEX-2T.</t> b 12.5 % Silver-stained polyacrylamide gel of the purified human ( lane 1 ) and porcine ( lane 3 ) GST-HMT fusion proteins used for the immunization of mice and the HMT and GST products resulting from cleavage with thrombin protease ( lanes 2 and 4 ). The sizes of molecular weight markers ( M ) are given on the left in kilodalton. c Sequence alignment and percent sequence identity of the HMT proteins from man, pig, mouse and rat obtained with the NCBI Constrained-based Multiple Alignment Tool ( www.ncbi.nlm.nih.gov/tools/cobalt/cobalt.cgi?link_loc=BlastHomeLink ). Residues identical in all four proteins are shaded black , residues identical in three proteins are shaded gray , and residues that have been shown to interact with histamine and S-adenosylhomocysteine in human HMT [ 7 ] are shaded red (indicated by plus symbol on top) and blue (indicated by hash on top), respectively. Antigenicity plots of human ( d ) and porcine ( e ) HMT produced with the BepiPred Linear Epitope Prediction Tool ( www.tools.immuneepitope.org/bcell ) [ 32 ] show similar predicted B-cell epitopes ( yellow peaks on top of the threshold line with reference to amino acid positions) for both protein sequences
    Prokaryotic Expression Vector Pgex 2t, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 87/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/prokaryotic expression vector pgex 2t/product/GE Healthcare
    Average 87 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    prokaryotic expression vector pgex 2t - by Bioz Stars, 2020-04
    87/100 stars
      Buy from Supplier

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    Recombinant HMT antigens used for immunizations. a Recombinant human and porcine GST-HMT fusion proteins cloned in the bacterial expression vector pGEX-2T. b 12.5 % Silver-stained polyacrylamide gel of the purified human ( lane 1 ) and porcine ( lane 3 ) GST-HMT fusion proteins used for the immunization of mice and the HMT and GST products resulting from cleavage with thrombin protease ( lanes 2 and 4 ). The sizes of molecular weight markers ( M ) are given on the left in kilodalton. c Sequence alignment and percent sequence identity of the HMT proteins from man, pig, mouse and rat obtained with the NCBI Constrained-based Multiple Alignment Tool ( www.ncbi.nlm.nih.gov/tools/cobalt/cobalt.cgi?link_loc=BlastHomeLink ). Residues identical in all four proteins are shaded black , residues identical in three proteins are shaded gray , and residues that have been shown to interact with histamine and S-adenosylhomocysteine in human HMT [ 7 ] are shaded red (indicated by plus symbol on top) and blue (indicated by hash on top), respectively. Antigenicity plots of human ( d ) and porcine ( e ) HMT produced with the BepiPred Linear Epitope Prediction Tool ( www.tools.immuneepitope.org/bcell ) [ 32 ] show similar predicted B-cell epitopes ( yellow peaks on top of the threshold line with reference to amino acid positions) for both protein sequences

    Journal: Inflammation Research

    Article Title: Monoclonal antibodies for human and porcine histamine N-methyltransferase (HMT) facilitate protein expression and localization studies

    doi: 10.1007/s00011-016-0987-1

    Figure Lengend Snippet: Recombinant HMT antigens used for immunizations. a Recombinant human and porcine GST-HMT fusion proteins cloned in the bacterial expression vector pGEX-2T. b 12.5 % Silver-stained polyacrylamide gel of the purified human ( lane 1 ) and porcine ( lane 3 ) GST-HMT fusion proteins used for the immunization of mice and the HMT and GST products resulting from cleavage with thrombin protease ( lanes 2 and 4 ). The sizes of molecular weight markers ( M ) are given on the left in kilodalton. c Sequence alignment and percent sequence identity of the HMT proteins from man, pig, mouse and rat obtained with the NCBI Constrained-based Multiple Alignment Tool ( www.ncbi.nlm.nih.gov/tools/cobalt/cobalt.cgi?link_loc=BlastHomeLink ). Residues identical in all four proteins are shaded black , residues identical in three proteins are shaded gray , and residues that have been shown to interact with histamine and S-adenosylhomocysteine in human HMT [ 7 ] are shaded red (indicated by plus symbol on top) and blue (indicated by hash on top), respectively. Antigenicity plots of human ( d ) and porcine ( e ) HMT produced with the BepiPred Linear Epitope Prediction Tool ( www.tools.immuneepitope.org/bcell ) [ 32 ] show similar predicted B-cell epitopes ( yellow peaks on top of the threshold line with reference to amino acid positions) for both protein sequences

    Article Snippet: Preparation of recombinant HMT proteins Full-length human and porcine HMT cDNAs [ , , ] were amplified by PCR with specific primers from total human and porcine kidney cDNA, respectively, and cloned in frame into the bacterial expression vector pGEX-2T (GE Healthcare, Vienna, Austria).

    Techniques: Recombinant, HMT Assay, Clone Assay, Expressing, Plasmid Preparation, Staining, Purification, Mouse Assay, Molecular Weight, Sequencing, Produced

    HNMT expression constructs. a Recombinant plasmids expressing GST-HNMT fusion proteins obtained by cloning different human HNMT cDNA fragments into the bacterial expression vectors pGEX-2T or pGEX-5X-1/-2, respectively. b 10% silver-stained SDS polyacrylamide gel and immunoblot with an anti-GST antibody of lysates prepared from bacteria harbouring plasmids pGEX-huHMT01-11 indicated on top of each lane . Migration positions of full-length fusion proteins are indicated by arrows and their expected sizes are listed in Table 1 . The sizes of molecular weight markers ( M ) are given on the left in kDa

    Journal: Inflammation Research

    Article Title: Mapping of the binding sites of human histamine N-methyltransferase (HNMT) monoclonal antibodies

    doi: 10.1007/s00011-017-1086-7

    Figure Lengend Snippet: HNMT expression constructs. a Recombinant plasmids expressing GST-HNMT fusion proteins obtained by cloning different human HNMT cDNA fragments into the bacterial expression vectors pGEX-2T or pGEX-5X-1/-2, respectively. b 10% silver-stained SDS polyacrylamide gel and immunoblot with an anti-GST antibody of lysates prepared from bacteria harbouring plasmids pGEX-huHMT01-11 indicated on top of each lane . Migration positions of full-length fusion proteins are indicated by arrows and their expected sizes are listed in Table 1 . The sizes of molecular weight markers ( M ) are given on the left in kDa

    Article Snippet: Preparation and expression of recombinant human HNMT protein fragments Full-length human HNMT cDNA [ , ] was amplified by PCR with specific primers from total human kidney cDNA and cloned in frame into the Bam HI site of the bacterial expression vector pGEX-2T (GE Healthcare, Vienna, Austria) to obtain plasmid pGEX-huHMT01 [ ].

    Techniques: Expressing, Construct, Recombinant, Clone Assay, Staining, Migration, Molecular Weight

    Schematic diagram of the expression vector pGEX-2T-Aβ(1–40). Factor Xa recognition site (IEGR) and Aβ(1–40) sequence were inserted into the Bam HI and Eco RI sites of pGEX-2T downstream from GST gene. The vector also had

    Journal: Protein expression and purification

    Article Title: Expression and purification of 15N- and 13C-isotope labeled 40-residue human Alzheimer's ?-amyloid peptide for NMR-based structural analysis

    doi: 10.1016/j.pep.2011.05.012

    Figure Lengend Snippet: Schematic diagram of the expression vector pGEX-2T-Aβ(1–40). Factor Xa recognition site (IEGR) and Aβ(1–40) sequence were inserted into the Bam HI and Eco RI sites of pGEX-2T downstream from GST gene. The vector also had

    Article Snippet: The expression vector pGEX-2T was purchased from GE Healthcare (Piscataway, NJ).

    Techniques: Expressing, Plasmid Preparation, Sequencing