amylose resin  (New England Biolabs)


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  • 99
    Name:
    Amylose Resin
    Description:
    Amylose Resin 100 ml
    Catalog Number:
    e8021l
    Price:
    1103
    Size:
    100 ml
    Category:
    Protein Purification Kit Components
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    Structured Review

    New England Biolabs amylose resin
    Amylose Resin
    Amylose Resin 100 ml
    https://www.bioz.com/result/amylose resin/product/New England Biolabs
    Average 99 stars, based on 736 article reviews
    Price from $9.99 to $1999.99
    amylose resin - by Bioz Stars, 2020-01
    99/100 stars

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    Related Articles

    Clone Assay:

    Article Title: The nuclear transcription factor Rtg1p functions as a cytosolic, post-transcriptional regulator in the methylotrophic yeast Pichia pastoris
    Article Snippet: The PCR product was digested with BamHI and PstI and cloned into pMAL-c2X vector. .. Recombinant proteins were expressed as MBP fusion proteins (PpRtgXp, PpRtgXp*, and ScRtg3p) or GST fusion proteins (PpRTG1 and ScRTG1) and purified using amylose resin (New England Biolabs) and GSH-agarose beads (G Biosciences), respectively, according to the manufacturers' instructions.

    Article Title: Multi-tiered pairing selectivity between E2 ubiquitin–conjugating enzymes and E3 ligases
    Article Snippet: .. The cloning of PUB22 into pMal-c2X vector (New England Biolabs), expression of the MBP-PUB22 in Escherichia coli Rosetta 2(DE3) pLysS and its purification by affinity chromatography using amylose resin (New England Biolabs) was previously described ( ). .. UBA1 and genes encoded the selected E2s ( UBC: 1, 5, 8, 9, 10, 11, 12, 17, 26, 28, 29, 30, 35 ) or MMZ4 were cloned into pENTR/D-TOPO (Invitrogen) entry clone and used for cloning into pDEST17 Gateway vector (Thermo Fisher Scientific) for His-tagged protein expression via LR reaction (Invitrogen).

    Article Title: A Tetratricopeptide Repeat Domain Protein has Profound Effects on Assembly of Periplasmic Flagella, Morphology, and Motility of the Lyme disease spirochete Borrelia burgdorferi
    Article Snippet: Subsequently, the codon optimized bb0236 was cloned in pMAL c5x expression vector (NEB Inc.) after removing the signal sequence (a.a. 1–20). .. The soluble fraction was incubated with 150 μl of amylose resin (NEB Inc.) at 4°C for overnight.

    Article Title: The Polycomb-Group Repressor MEDEA Attenuates Pathogen Defense
    Article Snippet: For construction of the MBP-Di19 recombinant fusion protein, Di19 sequences were cloned in the pMAL-p2X vector (New England Biolabs) at the C-terminal end of MBP between Eco RI and Bam HI restriction sites. .. MBP and MBP-Di19 were expressed in the Escherichia coli BL21 (DE3) strain and purified using amylose resin (New England Biolabs).

    Article Title: Cross-cleavage activity of Cas6b in crRNA processing of two different CRISPR-Cas systems in Methanosarcina mazei Gö1
    Article Snippet: Both fragments were TA-cloned into pCR4-TOPO (Invitrogen) resulting in pRS1078 ( cas6b -IB) and pRS1080 ( cas6b -IIIC) followed by subsequent cloning via Xmn I and Hind III from these plasmids to pMAL-cs (New England BioLabs). .. MBP-fusion proteins were purified from the supernatants by affinity chromatography with amylose resin according to the manufacturer’s instructions (New England BioLabs).

    Centrifugation:

    Article Title: The nuclear transcription factor Rtg1p functions as a cytosolic, post-transcriptional regulator in the methylotrophic yeast Pichia pastoris
    Article Snippet: Recombinant proteins were expressed as MBP fusion proteins (PpRtgXp, PpRtgXp*, and ScRtg3p) or GST fusion proteins (PpRTG1 and ScRTG1) and purified using amylose resin (New England Biolabs) and GSH-agarose beads (G Biosciences), respectively, according to the manufacturers' instructions. .. Cell lysates containing GST-Rtg1p were incubated with GSH-agarose beads at 4 °C for 1 h followed by brief centrifugation and washing with a buffer A containing 50 m m Tris (pH 8.0) and 150 m m NaCl.

    Article Title: Cross-cleavage activity of Cas6b in crRNA processing of two different CRISPR-Cas systems in Methanosarcina mazei Gö1
    Article Snippet: Expression of MBP-Cas6b-IB and MBP-Cas6b-IIIC was induced at a turbidity at 600 nm of 0.6 with 50 µM Isopropyl β-D-thiogalactoside for 3 h. Cells were harvested and disrupted in MBP-buffer (20 mM Tris/HCl pH 7.4, 200 mM NaCl, 1 mM EDTA) with a French pressure cell followed by centrifugation. .. MBP-fusion proteins were purified from the supernatants by affinity chromatography with amylose resin according to the manufacturer’s instructions (New England BioLabs).

    Article Title: A bifunctional role for the UHRF1 UBL domain in the control of hemi-methylated DNA-dependent histone ubiquitylation
    Article Snippet: Insoluble debris was pelleted by centrifugation at 17,500 rpm for 30 min to 1 hour at 4 °C. .. Alternatively, the His6 -MBP tag was removed with a combination of recapturing the tag on Ni-NTA resin after dialysis into 25 mM Tris-HCl pH 7.6, 200 mM NaCl, and 5 mM imidazole, and passage through amylose resin (New England BioLabs, cat# E8021S).

    Expressing:

    Article Title: The nuclear transcription factor Rtg1p functions as a cytosolic, post-transcriptional regulator in the methylotrophic yeast Pichia pastoris
    Article Snippet: Paragraph title: Expression of recombinant S. cerevisiae and P. pastoris Rtg proteins in E. coli and study of protein–protein interactions ... Recombinant proteins were expressed as MBP fusion proteins (PpRtgXp, PpRtgXp*, and ScRtg3p) or GST fusion proteins (PpRTG1 and ScRTG1) and purified using amylose resin (New England Biolabs) and GSH-agarose beads (G Biosciences), respectively, according to the manufacturers' instructions.

    Article Title: Multi-tiered pairing selectivity between E2 ubiquitin–conjugating enzymes and E3 ligases
    Article Snippet: .. The cloning of PUB22 into pMal-c2X vector (New England Biolabs), expression of the MBP-PUB22 in Escherichia coli Rosetta 2(DE3) pLysS and its purification by affinity chromatography using amylose resin (New England Biolabs) was previously described ( ). .. UBA1 and genes encoded the selected E2s ( UBC: 1, 5, 8, 9, 10, 11, 12, 17, 26, 28, 29, 30, 35 ) or MMZ4 were cloned into pENTR/D-TOPO (Invitrogen) entry clone and used for cloning into pDEST17 Gateway vector (Thermo Fisher Scientific) for His-tagged protein expression via LR reaction (Invitrogen).

    Article Title: A Tetratricopeptide Repeat Domain Protein has Profound Effects on Assembly of Periplasmic Flagella, Morphology, and Motility of the Lyme disease spirochete Borrelia burgdorferi
    Article Snippet: Subsequently, the codon optimized bb0236 was cloned in pMAL c5x expression vector (NEB Inc.) after removing the signal sequence (a.a. 1–20). .. The soluble fraction was incubated with 150 μl of amylose resin (NEB Inc.) at 4°C for overnight.

    Article Title: Cross-cleavage activity of Cas6b in crRNA processing of two different CRISPR-Cas systems in Methanosarcina mazei Gö1
    Article Snippet: Paragraph title: Heterologous expression of proteins in E. coli and purification ... MBP-fusion proteins were purified from the supernatants by affinity chromatography with amylose resin according to the manufacturer’s instructions (New England BioLabs).

    Article Title: A bifunctional role for the UHRF1 UBL domain in the control of hemi-methylated DNA-dependent histone ubiquitylation
    Article Snippet: Paragraph title: Protein Expression and Purification ... Alternatively, the His6 -MBP tag was removed with a combination of recapturing the tag on Ni-NTA resin after dialysis into 25 mM Tris-HCl pH 7.6, 200 mM NaCl, and 5 mM imidazole, and passage through amylose resin (New England BioLabs, cat# E8021S).

    Synthesized:

    Article Title: A Tetratricopeptide Repeat Domain Protein has Profound Effects on Assembly of Periplasmic Flagella, Morphology, and Motility of the Lyme disease spirochete Borrelia burgdorferi
    Article Snippet: To recombinantly express bb0236 in E. coli , the gene was synthesized after codon optimization. .. The soluble fraction was incubated with 150 μl of amylose resin (NEB Inc.) at 4°C for overnight.

    Construct:

    Article Title: The nuclear transcription factor Rtg1p functions as a cytosolic, post-transcriptional regulator in the methylotrophic yeast Pichia pastoris
    Article Snippet: The PCR product was digested with BamHI and PstI and cloned into pMAL-c2X vector. pMAL-PpRTGX * expression plasmid, expressing a chimera of PpRTGX and ScRTG3, was constructed by amplifying PpRTGX gene from GS115 genomic DNA and ScRTG3 gene encoding amino acids 345–486 from S. cerevisiae BY4741 by PCR using the following primer pairs: 5′-CGC GGATCC AATGGCACAAGATGCTCCCTTTG-3′ and 5′-CGTGCTTGTATTTCTAAAATTTCGGCCAGGTAGTTCGGAACGTCAAGGAAATTATTACCCGTG-3′ and 5′-CACGGGTAATAATTTCCTTGACGTTCCGAACTACCTGGCCGAAATTTTAGAAATACAAGCACG-3′ and 5′-AAAA CTGCAG TTAGTTCGGAACGTCAAGG-3′, respectively. .. Recombinant proteins were expressed as MBP fusion proteins (PpRtgXp, PpRtgXp*, and ScRtg3p) or GST fusion proteins (PpRTG1 and ScRTG1) and purified using amylose resin (New England Biolabs) and GSH-agarose beads (G Biosciences), respectively, according to the manufacturers' instructions.

    SYBR Green Assay:

    Article Title: Structural Basis for Influenza Virus NS1 Protein Block of mRNA Nuclear Export
    Article Snippet: .. 50xAdvantage Polymerase mix (Clontech (EMD), 639202); dNTP’s (Clontech (EMD), 639125); BamHI-HF (New England Biolabs (NEB), R3136T); NotI (New England Biolabs (NEB), R0189S); SmaI (New England Biolabs (NEB), R0141); XhoI (New England Biolabs (NEB), R0146S); T4 DNA Ligase (New England Biolabs (NEB), M0202); QIAquick Gel Extraction Kit (Qiagen, 28704); NEB® 5-alpha Competent E. coli (New England Biolabs (NEB), ); Rosetta (DE3) Competent Cells (EMD Millipore, 70954); SOC Outgrowth Medium (New England Biolabs (NEB), B9020); QIAprep Spin Miniprep Kit (250) (Qiagen, 27106); Lipofectamine® RNAiMAX Transfection Reagent (Thermo Fisher Scientific, 13778150); RNeasy Plus Mini Kit (Qiagen, 74134); Random Hexamers (50 μM) (Thermo Fisher Scientific, N8080127); Protector RNase Inhibitor (Roche, 03335402001); SuperScript II Reverse Transcriptase (Thermo Fisher Scientific, 18064014); LIGHTCYCLER 480 SYBR GREEN I MASTER (Roche, 04707516001); LightCycler® 480 Multiwell Plate 96, White (Roche, 04729692001); NE-PER™ Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific, 78833); Complete EDTA-free protease inhibitor tablets (Sigma-Aldrich, 11873580001); TransIT-X2® Dynamic Delivery System (Mirus Bio, MIR6000); SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific, 34096); L-Glutathione reduced (Sigma-Aldrich, G4251–25G); Ampicillin (Sigma-Aldrich, A-9518); Kanamycin Monosulfate (Gold Biotechnology, K-120–10); IPTG (Gold Biotechnology, I2481C50); Imidazole (Sigma-Aldrich, 56750); PMSF (RPI, ); Aprotinin (Santa Cruz Biotechnology, sc-3595); Leupeptin (Santa Cruz Biotechnology, sc295358); Pepstatin A (Thermo Fisher Scientific, ); Glutathione Sepharose 4B (GE Healthcare, 17–0756-01); Amylose Resin (New England Biolabs (NEB), E8021S); Ni-NTA Agarose (Qiagen, 30210); Mono Q 5/50 GL (GE Healthcare, 17-5166-01); HiTrap SP HP (GE Healthcare, 17-1151-01); Superdex 200 HR 10/30 (GE Healthcare, 17-1088-01); EasyTag EXPRESS35 S Protein Labeling Mix, [35S]-, 7mCi (PerkinElmer, NEG772007MC); T7 RiboMAX™ Express Large Scale RNA Production System (Promega, P1320); Protein G Sepharose® 4 Fast Flow (GE Healthcare, 17-0618-01); Hoechst 33258 (Molecular Probes/Life Technologies); M mRNA probes (Biosearch Technologies) ; ProLong Gold antifade reagent (Life Technologies, ). ..

    Incubation:

    Article Title: The nuclear transcription factor Rtg1p functions as a cytosolic, post-transcriptional regulator in the methylotrophic yeast Pichia pastoris
    Article Snippet: Recombinant proteins were expressed as MBP fusion proteins (PpRtgXp, PpRtgXp*, and ScRtg3p) or GST fusion proteins (PpRTG1 and ScRTG1) and purified using amylose resin (New England Biolabs) and GSH-agarose beads (G Biosciences), respectively, according to the manufacturers' instructions. .. Cell lysates containing GST-Rtg1p were incubated with GSH-agarose beads at 4 °C for 1 h followed by brief centrifugation and washing with a buffer A containing 50 m m Tris (pH 8.0) and 150 m m NaCl.

    Article Title: Importance of the Conserved Carboxyl-Terminal CNOT1 Binding Domain to Tristetraprolin Activity In Vivo
    Article Snippet: .. Cells were lysed as described above, and supernatants were incubated with amylose resin (New England Biolabs) and eluted with 40 mM maltose. ..

    Article Title: A Tetratricopeptide Repeat Domain Protein has Profound Effects on Assembly of Periplasmic Flagella, Morphology, and Motility of the Lyme disease spirochete Borrelia burgdorferi
    Article Snippet: .. The soluble fraction was incubated with 150 μl of amylose resin (NEB Inc.) at 4°C for overnight. ..

    Article Title: Inheritance of CENP-A nucleosomes during DNA replication requires HJURP
    Article Snippet: .. Thirty microliters of 50 μM MBP-His-MCM243−160 was incubated with 20 μl of amylose resin (New England Biolabs) in P300 buffer (NaP pH 7.0, 300mM NaCl). ..

    Article Title: Protein interaction mapping identifies RBBP6 as a negative regulator of Ebola virus replication
    Article Snippet: Amylose resin (NEB) was equilibrated with buffer containing 20 mM Tris pH 7.5, 150 mM NaCl, and 5 mM β-ME. .. Equilibrated resin was incubated with cell lysate for 15 min at 4°C, washed, and resuspended in buffer.

    Article Title: Dual Role of the C-Terminal Domain in Osmosensing by Bacterial Osmolyte Transporter ProP
    Article Snippet: .. The culture was incubated at 37°C with shaking (200 rotations per minute) for 1–1.5 h. Isopropyl β -D-1-thiogalactopyranoside (1 mM) was added, and the incubation was continued at 25°C for 4 h. The proteins were purified from periplasmic extracts by affinity chromatography using Amylose resin (catalog number E80215; New England Biolabs) according to the vendor’s instructions for the purification of periplasmic proteins. .. The molecular weights of the intact, purified proteins were determined using an Agilent UHD 6530 Q-ToF electrospray mass spectrometer (Agilent, Santa Clara, CA) at the Mass Spectrometry Facility of the Advanced Analysis Centre, University of Guelph.

    Article Title: A Hippo-like signalling pathway controls tracheal morphogenesis in Drosophila melanogaster
    Article Snippet: Recombinant Mal-fusion proteins were expressed in E. coli BL21(DE3) at 30°C and purified using amylose resin (E8021, New England BioLabs) as described ( ). .. Immunopurified proteins were washed twice with kinase buffer (50 mM Hepes pH 7.4, 10 mM MgCl2 , 2.5 mM beta-glycerophosphate, 1 mM EGTA, 1 mM Na3 VO4 , 1 mM NaF, 0.01 mM DTT), before kinase reactions were performed as follows: per reaction, 200 ng of Mal-fusion proteins were incubated at 30°C for 30 minutes in 20 μl of kinase buffer containing 100 μM ATP in the absence or presence of immunopurified Tao-1, TAO1 or GCKIII or GST-tagged kinases (100 ng per reaction).

    Mass Spectrometry:

    Article Title: Dual Role of the C-Terminal Domain in Osmosensing by Bacterial Osmolyte Transporter ProP
    Article Snippet: The culture was incubated at 37°C with shaking (200 rotations per minute) for 1–1.5 h. Isopropyl β -D-1-thiogalactopyranoside (1 mM) was added, and the incubation was continued at 25°C for 4 h. The proteins were purified from periplasmic extracts by affinity chromatography using Amylose resin (catalog number E80215; New England Biolabs) according to the vendor’s instructions for the purification of periplasmic proteins. .. The molecular weights of the intact, purified proteins were determined using an Agilent UHD 6530 Q-ToF electrospray mass spectrometer (Agilent, Santa Clara, CA) at the Mass Spectrometry Facility of the Advanced Analysis Centre, University of Guelph.

    Transformation Assay:

    Article Title: The nuclear transcription factor Rtg1p functions as a cytosolic, post-transcriptional regulator in the methylotrophic yeast Pichia pastoris
    Article Snippet: Recombinant plasmids were transformed into E. coli BL21 (DE3) strain. .. Recombinant proteins were expressed as MBP fusion proteins (PpRtgXp, PpRtgXp*, and ScRtg3p) or GST fusion proteins (PpRTG1 and ScRTG1) and purified using amylose resin (New England Biolabs) and GSH-agarose beads (G Biosciences), respectively, according to the manufacturers' instructions.

    Article Title: Cross-cleavage activity of Cas6b in crRNA processing of two different CRISPR-Cas systems in Methanosarcina mazei Gö1
    Article Snippet: The received plasmids were designated pRS1079 ( cas6b -IB) and pRS1081 ( cas6b -IIIC) and were each transformed into E. coli BL21-CodonPlus®-RIL (Stratagene) for the heterologous protein expression as MBP (maltose-binding protein) fusion proteins. .. MBP-fusion proteins were purified from the supernatants by affinity chromatography with amylose resin according to the manufacturer’s instructions (New England BioLabs).

    Flow Cytometry:

    Article Title: Structural Basis for Influenza Virus NS1 Protein Block of mRNA Nuclear Export
    Article Snippet: .. 50xAdvantage Polymerase mix (Clontech (EMD), 639202); dNTP’s (Clontech (EMD), 639125); BamHI-HF (New England Biolabs (NEB), R3136T); NotI (New England Biolabs (NEB), R0189S); SmaI (New England Biolabs (NEB), R0141); XhoI (New England Biolabs (NEB), R0146S); T4 DNA Ligase (New England Biolabs (NEB), M0202); QIAquick Gel Extraction Kit (Qiagen, 28704); NEB® 5-alpha Competent E. coli (New England Biolabs (NEB), ); Rosetta (DE3) Competent Cells (EMD Millipore, 70954); SOC Outgrowth Medium (New England Biolabs (NEB), B9020); QIAprep Spin Miniprep Kit (250) (Qiagen, 27106); Lipofectamine® RNAiMAX Transfection Reagent (Thermo Fisher Scientific, 13778150); RNeasy Plus Mini Kit (Qiagen, 74134); Random Hexamers (50 μM) (Thermo Fisher Scientific, N8080127); Protector RNase Inhibitor (Roche, 03335402001); SuperScript II Reverse Transcriptase (Thermo Fisher Scientific, 18064014); LIGHTCYCLER 480 SYBR GREEN I MASTER (Roche, 04707516001); LightCycler® 480 Multiwell Plate 96, White (Roche, 04729692001); NE-PER™ Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific, 78833); Complete EDTA-free protease inhibitor tablets (Sigma-Aldrich, 11873580001); TransIT-X2® Dynamic Delivery System (Mirus Bio, MIR6000); SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific, 34096); L-Glutathione reduced (Sigma-Aldrich, G4251–25G); Ampicillin (Sigma-Aldrich, A-9518); Kanamycin Monosulfate (Gold Biotechnology, K-120–10); IPTG (Gold Biotechnology, I2481C50); Imidazole (Sigma-Aldrich, 56750); PMSF (RPI, ); Aprotinin (Santa Cruz Biotechnology, sc-3595); Leupeptin (Santa Cruz Biotechnology, sc295358); Pepstatin A (Thermo Fisher Scientific, ); Glutathione Sepharose 4B (GE Healthcare, 17–0756-01); Amylose Resin (New England Biolabs (NEB), E8021S); Ni-NTA Agarose (Qiagen, 30210); Mono Q 5/50 GL (GE Healthcare, 17-5166-01); HiTrap SP HP (GE Healthcare, 17-1151-01); Superdex 200 HR 10/30 (GE Healthcare, 17-1088-01); EasyTag EXPRESS35 S Protein Labeling Mix, [35S]-, 7mCi (PerkinElmer, NEG772007MC); T7 RiboMAX™ Express Large Scale RNA Production System (Promega, P1320); Protein G Sepharose® 4 Fast Flow (GE Healthcare, 17-0618-01); Hoechst 33258 (Molecular Probes/Life Technologies); M mRNA probes (Biosearch Technologies) ; ProLong Gold antifade reagent (Life Technologies, ). ..

    Chromatography:

    Article Title: Cross-cleavage activity of Cas6b in crRNA processing of two different CRISPR-Cas systems in Methanosarcina mazei Gö1
    Article Snippet: MBP-fusion proteins were purified from the supernatants by affinity chromatography with amylose resin according to the manufacturer’s instructions (New England BioLabs). .. The purified proteins were dialyzed against 10 mM Tris/HCL pH 7.6, followed by subsequent purification by anion-exchange chromatography using a Q-Seharose FF (XK26, Pharmacia Biotech) as described before [ ].

    Concentration Assay:

    Article Title: Importance of the Conserved Carboxyl-Terminal CNOT1 Binding Domain to Tristetraprolin Activity In Vivo
    Article Snippet: After concentration using a 3,000 MWCO Vivaspin 20 centrifugal device, purified protein was flash frozen in liquid nitrogen and stored at −80°C. .. Cells were lysed as described above, and supernatants were incubated with amylose resin (New England Biolabs) and eluted with 40 mM maltose.

    Article Title: A bifunctional role for the UHRF1 UBL domain in the control of hemi-methylated DNA-dependent histone ubiquitylation
    Article Snippet: Phenylmethylsulfonyl fluoride (PMSF; Sigma, Cat# 329–98-6) was added to a final concentration 1 mM to the resuspended cell pellet immediately prior to cell lyses, which was performed using a short sonication, followed by homogenization or by two passages through a French pressure cell. .. Alternatively, the His6 -MBP tag was removed with a combination of recapturing the tag on Ni-NTA resin after dialysis into 25 mM Tris-HCl pH 7.6, 200 mM NaCl, and 5 mM imidazole, and passage through amylose resin (New England BioLabs, cat# E8021S).

    Protease Inhibitor:

    Article Title: NRDE2 negatively regulates exosome functions by inhibiting MTR4 recruitment and exosome interaction
    Article Snippet: .. For each pull-down reaction, 8 μg of MBP-tagged proteins and 8 μg of His-tagged proteins were added to 20 μL of Amylose Resin (New England BioLabs) in pull-down buffer (1× PBS, 0.1% Triton, 0.2 mM PMSF, protease inhibitor). ..

    Article Title: Structural Basis for Influenza Virus NS1 Protein Block of mRNA Nuclear Export
    Article Snippet: .. 50xAdvantage Polymerase mix (Clontech (EMD), 639202); dNTP’s (Clontech (EMD), 639125); BamHI-HF (New England Biolabs (NEB), R3136T); NotI (New England Biolabs (NEB), R0189S); SmaI (New England Biolabs (NEB), R0141); XhoI (New England Biolabs (NEB), R0146S); T4 DNA Ligase (New England Biolabs (NEB), M0202); QIAquick Gel Extraction Kit (Qiagen, 28704); NEB® 5-alpha Competent E. coli (New England Biolabs (NEB), ); Rosetta (DE3) Competent Cells (EMD Millipore, 70954); SOC Outgrowth Medium (New England Biolabs (NEB), B9020); QIAprep Spin Miniprep Kit (250) (Qiagen, 27106); Lipofectamine® RNAiMAX Transfection Reagent (Thermo Fisher Scientific, 13778150); RNeasy Plus Mini Kit (Qiagen, 74134); Random Hexamers (50 μM) (Thermo Fisher Scientific, N8080127); Protector RNase Inhibitor (Roche, 03335402001); SuperScript II Reverse Transcriptase (Thermo Fisher Scientific, 18064014); LIGHTCYCLER 480 SYBR GREEN I MASTER (Roche, 04707516001); LightCycler® 480 Multiwell Plate 96, White (Roche, 04729692001); NE-PER™ Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific, 78833); Complete EDTA-free protease inhibitor tablets (Sigma-Aldrich, 11873580001); TransIT-X2® Dynamic Delivery System (Mirus Bio, MIR6000); SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific, 34096); L-Glutathione reduced (Sigma-Aldrich, G4251–25G); Ampicillin (Sigma-Aldrich, A-9518); Kanamycin Monosulfate (Gold Biotechnology, K-120–10); IPTG (Gold Biotechnology, I2481C50); Imidazole (Sigma-Aldrich, 56750); PMSF (RPI, ); Aprotinin (Santa Cruz Biotechnology, sc-3595); Leupeptin (Santa Cruz Biotechnology, sc295358); Pepstatin A (Thermo Fisher Scientific, ); Glutathione Sepharose 4B (GE Healthcare, 17–0756-01); Amylose Resin (New England Biolabs (NEB), E8021S); Ni-NTA Agarose (Qiagen, 30210); Mono Q 5/50 GL (GE Healthcare, 17-5166-01); HiTrap SP HP (GE Healthcare, 17-1151-01); Superdex 200 HR 10/30 (GE Healthcare, 17-1088-01); EasyTag EXPRESS35 S Protein Labeling Mix, [35S]-, 7mCi (PerkinElmer, NEG772007MC); T7 RiboMAX™ Express Large Scale RNA Production System (Promega, P1320); Protein G Sepharose® 4 Fast Flow (GE Healthcare, 17-0618-01); Hoechst 33258 (Molecular Probes/Life Technologies); M mRNA probes (Biosearch Technologies) ; ProLong Gold antifade reagent (Life Technologies, ). ..

    Generated:

    Article Title: AKT inhibition-mediated dephosphorylation of TFE3 promotes overactive autophagy independent of MTORC1 in cadmium-exposed bone mesenchymal stem cells
    Article Snippet: MBP-TFE3 and MBP-TFE3S565A proteins were expressed in bacterial BL21 (DE3) cells and purified with amylose resin (New England BioLabs, E8021S). .. All of the purified active AKT1, TFE3 enzyme and TFE3S565A proteins are generated by GenScript Corporation (Nanjing, China).

    Polymerase Chain Reaction:

    Article Title: The nuclear transcription factor Rtg1p functions as a cytosolic, post-transcriptional regulator in the methylotrophic yeast Pichia pastoris
    Article Snippet: The PCR product was digested with BamHI and PstI and cloned into pMAL-c2X vector. .. Recombinant proteins were expressed as MBP fusion proteins (PpRtgXp, PpRtgXp*, and ScRtg3p) or GST fusion proteins (PpRTG1 and ScRTG1) and purified using amylose resin (New England Biolabs) and GSH-agarose beads (G Biosciences), respectively, according to the manufacturers' instructions.

    Sonication:

    Article Title: The nuclear transcription factor Rtg1p functions as a cytosolic, post-transcriptional regulator in the methylotrophic yeast Pichia pastoris
    Article Snippet: Recombinant proteins were expressed as MBP fusion proteins (PpRtgXp, PpRtgXp*, and ScRtg3p) or GST fusion proteins (PpRTG1 and ScRTG1) and purified using amylose resin (New England Biolabs) and GSH-agarose beads (G Biosciences), respectively, according to the manufacturers' instructions. .. For the study of protein–protein interactions, E. coli cells were suspended in a buffer containing 50 m m Tris (pH 8.0), 300 m m NaCl, 10% glycerol, 10 m m β-Mercaptoethanol, and 1 m m PMSF and subjected to sonication in a Vibracell 750 (Sonics and Materials Inc., Newtown, CT).

    Article Title: A bifunctional role for the UHRF1 UBL domain in the control of hemi-methylated DNA-dependent histone ubiquitylation
    Article Snippet: Phenylmethylsulfonyl fluoride (PMSF; Sigma, Cat# 329–98-6) was added to a final concentration 1 mM to the resuspended cell pellet immediately prior to cell lyses, which was performed using a short sonication, followed by homogenization or by two passages through a French pressure cell. .. Alternatively, the His6 -MBP tag was removed with a combination of recapturing the tag on Ni-NTA resin after dialysis into 25 mM Tris-HCl pH 7.6, 200 mM NaCl, and 5 mM imidazole, and passage through amylose resin (New England BioLabs, cat# E8021S).

    Recombinant:

    Article Title: The nuclear transcription factor Rtg1p functions as a cytosolic, post-transcriptional regulator in the methylotrophic yeast Pichia pastoris
    Article Snippet: .. Recombinant proteins were expressed as MBP fusion proteins (PpRtgXp, PpRtgXp*, and ScRtg3p) or GST fusion proteins (PpRTG1 and ScRTG1) and purified using amylose resin (New England Biolabs) and GSH-agarose beads (G Biosciences), respectively, according to the manufacturers' instructions. .. For the study of protein–protein interactions, E. coli cells were suspended in a buffer containing 50 m m Tris (pH 8.0), 300 m m NaCl, 10% glycerol, 10 m m β-Mercaptoethanol, and 1 m m PMSF and subjected to sonication in a Vibracell 750 (Sonics and Materials Inc., Newtown, CT).

    Article Title: Multi-tiered pairing selectivity between E2 ubiquitin–conjugating enzymes and E3 ligases
    Article Snippet: Paragraph title: Site-directed mutagenesis and recombinant protein expression and purification ... The cloning of PUB22 into pMal-c2X vector (New England Biolabs), expression of the MBP-PUB22 in Escherichia coli Rosetta 2(DE3) pLysS and its purification by affinity chromatography using amylose resin (New England Biolabs) was previously described ( ).

    Article Title: The Polycomb-Group Repressor MEDEA Attenuates Pathogen Defense
    Article Snippet: For construction of the MBP-Di19 recombinant fusion protein, Di19 sequences were cloned in the pMAL-p2X vector (New England Biolabs) at the C-terminal end of MBP between Eco RI and Bam HI restriction sites. .. MBP and MBP-Di19 were expressed in the Escherichia coli BL21 (DE3) strain and purified using amylose resin (New England Biolabs).

    Article Title: Inheritance of CENP-A nucleosomes during DNA replication requires HJURP
    Article Snippet: Paragraph title: In vitro recombinant protein pull-downs ... Thirty microliters of 50 μM MBP-His-MCM243−160 was incubated with 20 μl of amylose resin (New England Biolabs) in P300 buffer (NaP pH 7.0, 300mM NaCl).

    Article Title: A Hippo-like signalling pathway controls tracheal morphogenesis in Drosophila melanogaster
    Article Snippet: .. Recombinant Mal-fusion proteins were expressed in E. coli BL21(DE3) at 30°C and purified using amylose resin (E8021, New England BioLabs) as described ( ). .. Recombinant full-length GST-MST1 (M9697; 07–116), GST-MST2 (S6573; 07–117), GST-MST3 (M9822; 07–118), GST-MST4 (M9947; 07–119) and GST-STK25 (SRP5087; 07–136) were from Sigma and Carna Biosciences, respectively.

    Article Title: AKT inhibition-mediated dephosphorylation of TFE3 promotes overactive autophagy independent of MTORC1 in cadmium-exposed bone mesenchymal stem cells
    Article Snippet: Paragraph title: Recombinant protein ... MBP-TFE3 and MBP-TFE3S565A proteins were expressed in bacterial BL21 (DE3) cells and purified with amylose resin (New England BioLabs, E8021S).

    Pull Down Assay:

    Article Title: A Tetratricopeptide Repeat Domain Protein has Profound Effects on Assembly of Periplasmic Flagella, Morphology, and Motility of the Lyme disease spirochete Borrelia burgdorferi
    Article Snippet: Paragraph title: Pull-down assay and Far-western. ... The soluble fraction was incubated with 150 μl of amylose resin (NEB Inc.) at 4°C for overnight.

    Mutagenesis:

    Article Title: Multi-tiered pairing selectivity between E2 ubiquitin–conjugating enzymes and E3 ligases
    Article Snippet: Paragraph title: Site-directed mutagenesis and recombinant protein expression and purification ... The cloning of PUB22 into pMal-c2X vector (New England Biolabs), expression of the MBP-PUB22 in Escherichia coli Rosetta 2(DE3) pLysS and its purification by affinity chromatography using amylose resin (New England Biolabs) was previously described ( ).

    Article Title: Importance of the Conserved Carboxyl-Terminal CNOT1 Binding Domain to Tristetraprolin Activity In Vivo
    Article Snippet: The human TTP TZF domain fusion peptide was expressed in BL21(DE3) cells (Millipore-Sigma) after induction with 0.1 mM IPTG for 16 h at 20°C and was purified as described above for full-length and mutant TTP proteins, with the exception that the affinity tag was removed following elution from the HisTrap column by incubation with tobacco etch virus (TEV) protease (vector kindly supplied by Traci T. Hall, NIEHS) for 16 h at 4°C in the presence of 2 mM DTT. .. Cells were lysed as described above, and supernatants were incubated with amylose resin (New England Biolabs) and eluted with 40 mM maltose.

    Isolation:

    Article Title: Importance of the Conserved Carboxyl-Terminal CNOT1 Binding Domain to Tristetraprolin Activity In Vivo
    Article Snippet: Isolated TZF domains were further purified using a Superdex S200 column in 20 mM HEPES, pH 7.5, 100 mM KCl, 25 μM ZnSO4 , 2 mM 2-mercaptoethanol, and 5% glycerol. .. Cells were lysed as described above, and supernatants were incubated with amylose resin (New England Biolabs) and eluted with 40 mM maltose.

    Article Title: A bifunctional role for the UHRF1 UBL domain in the control of hemi-methylated DNA-dependent histone ubiquitylation
    Article Snippet: When removing the MBP tag from the isolated UBL domains and mutants, the UBL domain did not bind to the column at 50 mM NaCl. .. Alternatively, the His6 -MBP tag was removed with a combination of recapturing the tag on Ni-NTA resin after dialysis into 25 mM Tris-HCl pH 7.6, 200 mM NaCl, and 5 mM imidazole, and passage through amylose resin (New England BioLabs, cat# E8021S).

    Transfection:

    Article Title: Structural Basis for Influenza Virus NS1 Protein Block of mRNA Nuclear Export
    Article Snippet: .. 50xAdvantage Polymerase mix (Clontech (EMD), 639202); dNTP’s (Clontech (EMD), 639125); BamHI-HF (New England Biolabs (NEB), R3136T); NotI (New England Biolabs (NEB), R0189S); SmaI (New England Biolabs (NEB), R0141); XhoI (New England Biolabs (NEB), R0146S); T4 DNA Ligase (New England Biolabs (NEB), M0202); QIAquick Gel Extraction Kit (Qiagen, 28704); NEB® 5-alpha Competent E. coli (New England Biolabs (NEB), ); Rosetta (DE3) Competent Cells (EMD Millipore, 70954); SOC Outgrowth Medium (New England Biolabs (NEB), B9020); QIAprep Spin Miniprep Kit (250) (Qiagen, 27106); Lipofectamine® RNAiMAX Transfection Reagent (Thermo Fisher Scientific, 13778150); RNeasy Plus Mini Kit (Qiagen, 74134); Random Hexamers (50 μM) (Thermo Fisher Scientific, N8080127); Protector RNase Inhibitor (Roche, 03335402001); SuperScript II Reverse Transcriptase (Thermo Fisher Scientific, 18064014); LIGHTCYCLER 480 SYBR GREEN I MASTER (Roche, 04707516001); LightCycler® 480 Multiwell Plate 96, White (Roche, 04729692001); NE-PER™ Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific, 78833); Complete EDTA-free protease inhibitor tablets (Sigma-Aldrich, 11873580001); TransIT-X2® Dynamic Delivery System (Mirus Bio, MIR6000); SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific, 34096); L-Glutathione reduced (Sigma-Aldrich, G4251–25G); Ampicillin (Sigma-Aldrich, A-9518); Kanamycin Monosulfate (Gold Biotechnology, K-120–10); IPTG (Gold Biotechnology, I2481C50); Imidazole (Sigma-Aldrich, 56750); PMSF (RPI, ); Aprotinin (Santa Cruz Biotechnology, sc-3595); Leupeptin (Santa Cruz Biotechnology, sc295358); Pepstatin A (Thermo Fisher Scientific, ); Glutathione Sepharose 4B (GE Healthcare, 17–0756-01); Amylose Resin (New England Biolabs (NEB), E8021S); Ni-NTA Agarose (Qiagen, 30210); Mono Q 5/50 GL (GE Healthcare, 17-5166-01); HiTrap SP HP (GE Healthcare, 17-1151-01); Superdex 200 HR 10/30 (GE Healthcare, 17-1088-01); EasyTag EXPRESS35 S Protein Labeling Mix, [35S]-, 7mCi (PerkinElmer, NEG772007MC); T7 RiboMAX™ Express Large Scale RNA Production System (Promega, P1320); Protein G Sepharose® 4 Fast Flow (GE Healthcare, 17-0618-01); Hoechst 33258 (Molecular Probes/Life Technologies); M mRNA probes (Biosearch Technologies) ; ProLong Gold antifade reagent (Life Technologies, ). ..

    Article Title: A Hippo-like signalling pathway controls tracheal morphogenesis in Drosophila melanogaster
    Article Snippet: Recombinant Mal-fusion proteins were expressed in E. coli BL21(DE3) at 30°C and purified using amylose resin (E8021, New England BioLabs) as described ( ). .. To produce immunopurified full-length HA-tagged Tao-1 or TAO1 kinase version, D. melanogaster S2R+ or human HEK293 cells were transfected with pAW_HA-Tao1 or pcDNA3_HA-TAO1 and processed for immunoprecipitation (IP) using anti-HA antibody and stringent IP conditions as described ( ).

    Labeling:

    Article Title: Structural Basis for Influenza Virus NS1 Protein Block of mRNA Nuclear Export
    Article Snippet: .. 50xAdvantage Polymerase mix (Clontech (EMD), 639202); dNTP’s (Clontech (EMD), 639125); BamHI-HF (New England Biolabs (NEB), R3136T); NotI (New England Biolabs (NEB), R0189S); SmaI (New England Biolabs (NEB), R0141); XhoI (New England Biolabs (NEB), R0146S); T4 DNA Ligase (New England Biolabs (NEB), M0202); QIAquick Gel Extraction Kit (Qiagen, 28704); NEB® 5-alpha Competent E. coli (New England Biolabs (NEB), ); Rosetta (DE3) Competent Cells (EMD Millipore, 70954); SOC Outgrowth Medium (New England Biolabs (NEB), B9020); QIAprep Spin Miniprep Kit (250) (Qiagen, 27106); Lipofectamine® RNAiMAX Transfection Reagent (Thermo Fisher Scientific, 13778150); RNeasy Plus Mini Kit (Qiagen, 74134); Random Hexamers (50 μM) (Thermo Fisher Scientific, N8080127); Protector RNase Inhibitor (Roche, 03335402001); SuperScript II Reverse Transcriptase (Thermo Fisher Scientific, 18064014); LIGHTCYCLER 480 SYBR GREEN I MASTER (Roche, 04707516001); LightCycler® 480 Multiwell Plate 96, White (Roche, 04729692001); NE-PER™ Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific, 78833); Complete EDTA-free protease inhibitor tablets (Sigma-Aldrich, 11873580001); TransIT-X2® Dynamic Delivery System (Mirus Bio, MIR6000); SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific, 34096); L-Glutathione reduced (Sigma-Aldrich, G4251–25G); Ampicillin (Sigma-Aldrich, A-9518); Kanamycin Monosulfate (Gold Biotechnology, K-120–10); IPTG (Gold Biotechnology, I2481C50); Imidazole (Sigma-Aldrich, 56750); PMSF (RPI, ); Aprotinin (Santa Cruz Biotechnology, sc-3595); Leupeptin (Santa Cruz Biotechnology, sc295358); Pepstatin A (Thermo Fisher Scientific, ); Glutathione Sepharose 4B (GE Healthcare, 17–0756-01); Amylose Resin (New England Biolabs (NEB), E8021S); Ni-NTA Agarose (Qiagen, 30210); Mono Q 5/50 GL (GE Healthcare, 17-5166-01); HiTrap SP HP (GE Healthcare, 17-1151-01); Superdex 200 HR 10/30 (GE Healthcare, 17-1088-01); EasyTag EXPRESS35 S Protein Labeling Mix, [35S]-, 7mCi (PerkinElmer, NEG772007MC); T7 RiboMAX™ Express Large Scale RNA Production System (Promega, P1320); Protein G Sepharose® 4 Fast Flow (GE Healthcare, 17-0618-01); Hoechst 33258 (Molecular Probes/Life Technologies); M mRNA probes (Biosearch Technologies) ; ProLong Gold antifade reagent (Life Technologies, ). ..

    Purification:

    Article Title: The nuclear transcription factor Rtg1p functions as a cytosolic, post-transcriptional regulator in the methylotrophic yeast Pichia pastoris
    Article Snippet: .. Recombinant proteins were expressed as MBP fusion proteins (PpRtgXp, PpRtgXp*, and ScRtg3p) or GST fusion proteins (PpRTG1 and ScRTG1) and purified using amylose resin (New England Biolabs) and GSH-agarose beads (G Biosciences), respectively, according to the manufacturers' instructions. .. For the study of protein–protein interactions, E. coli cells were suspended in a buffer containing 50 m m Tris (pH 8.0), 300 m m NaCl, 10% glycerol, 10 m m β-Mercaptoethanol, and 1 m m PMSF and subjected to sonication in a Vibracell 750 (Sonics and Materials Inc., Newtown, CT).

    Article Title: Multi-tiered pairing selectivity between E2 ubiquitin–conjugating enzymes and E3 ligases
    Article Snippet: .. The cloning of PUB22 into pMal-c2X vector (New England Biolabs), expression of the MBP-PUB22 in Escherichia coli Rosetta 2(DE3) pLysS and its purification by affinity chromatography using amylose resin (New England Biolabs) was previously described ( ). .. UBA1 and genes encoded the selected E2s ( UBC: 1, 5, 8, 9, 10, 11, 12, 17, 26, 28, 29, 30, 35 ) or MMZ4 were cloned into pENTR/D-TOPO (Invitrogen) entry clone and used for cloning into pDEST17 Gateway vector (Thermo Fisher Scientific) for His-tagged protein expression via LR reaction (Invitrogen).

    Article Title: Importance of the Conserved Carboxyl-Terminal CNOT1 Binding Domain to Tristetraprolin Activity In Vivo
    Article Snippet: After concentration using a 3,000 MWCO Vivaspin 20 centrifugal device, purified protein was flash frozen in liquid nitrogen and stored at −80°C. .. Cells were lysed as described above, and supernatants were incubated with amylose resin (New England Biolabs) and eluted with 40 mM maltose.

    Article Title: The Polycomb-Group Repressor MEDEA Attenuates Pathogen Defense
    Article Snippet: .. MBP and MBP-Di19 were expressed in the Escherichia coli BL21 (DE3) strain and purified using amylose resin (New England Biolabs). .. For the binding assay, 400 ng, 1 µg, 1.6 µg and 2 µg of MBP or MBP Di19 was used.

    Article Title: Cross-cleavage activity of Cas6b in crRNA processing of two different CRISPR-Cas systems in Methanosarcina mazei Gö1
    Article Snippet: .. MBP-fusion proteins were purified from the supernatants by affinity chromatography with amylose resin according to the manufacturer’s instructions (New England BioLabs). .. The purified proteins were dialyzed against 10 mM Tris/HCL pH 7.6, followed by subsequent purification by anion-exchange chromatography using a Q-Seharose FF (XK26, Pharmacia Biotech) as described before [ ].

    Article Title: A bifunctional role for the UHRF1 UBL domain in the control of hemi-methylated DNA-dependent histone ubiquitylation
    Article Snippet: Paragraph title: Protein Expression and Purification ... Alternatively, the His6 -MBP tag was removed with a combination of recapturing the tag on Ni-NTA resin after dialysis into 25 mM Tris-HCl pH 7.6, 200 mM NaCl, and 5 mM imidazole, and passage through amylose resin (New England BioLabs, cat# E8021S).

    Article Title: Protein interaction mapping identifies RBBP6 as a negative regulator of Ebola virus replication
    Article Snippet: Amylose resin (NEB) was equilibrated with buffer containing 20 mM Tris pH 7.5, 150 mM NaCl, and 5 mM β-ME. .. Purified MBP-VP30s were then applied to the resin and allowed to incubate for 20 min.

    Article Title: Dual Role of the C-Terminal Domain in Osmosensing by Bacterial Osmolyte Transporter ProP
    Article Snippet: .. The culture was incubated at 37°C with shaking (200 rotations per minute) for 1–1.5 h. Isopropyl β -D-1-thiogalactopyranoside (1 mM) was added, and the incubation was continued at 25°C for 4 h. The proteins were purified from periplasmic extracts by affinity chromatography using Amylose resin (catalog number E80215; New England Biolabs) according to the vendor’s instructions for the purification of periplasmic proteins. .. The molecular weights of the intact, purified proteins were determined using an Agilent UHD 6530 Q-ToF electrospray mass spectrometer (Agilent, Santa Clara, CA) at the Mass Spectrometry Facility of the Advanced Analysis Centre, University of Guelph.

    Article Title: A Hippo-like signalling pathway controls tracheal morphogenesis in Drosophila melanogaster
    Article Snippet: .. Recombinant Mal-fusion proteins were expressed in E. coli BL21(DE3) at 30°C and purified using amylose resin (E8021, New England BioLabs) as described ( ). .. Recombinant full-length GST-MST1 (M9697; 07–116), GST-MST2 (S6573; 07–117), GST-MST3 (M9822; 07–118), GST-MST4 (M9947; 07–119) and GST-STK25 (SRP5087; 07–136) were from Sigma and Carna Biosciences, respectively.

    Article Title: AKT inhibition-mediated dephosphorylation of TFE3 promotes overactive autophagy independent of MTORC1 in cadmium-exposed bone mesenchymal stem cells
    Article Snippet: .. MBP-TFE3 and MBP-TFE3S565A proteins were expressed in bacterial BL21 (DE3) cells and purified with amylose resin (New England BioLabs, E8021S). .. All of the purified active AKT1, TFE3 enzyme and TFE3S565A proteins are generated by GenScript Corporation (Nanjing, China).

    Protein Purification:

    Article Title: Dual Role of the C-Terminal Domain in Osmosensing by Bacterial Osmolyte Transporter ProP
    Article Snippet: Paragraph title: Protein purification and liposome binding assays ... The culture was incubated at 37°C with shaking (200 rotations per minute) for 1–1.5 h. Isopropyl β -D-1-thiogalactopyranoside (1 mM) was added, and the incubation was continued at 25°C for 4 h. The proteins were purified from periplasmic extracts by affinity chromatography using Amylose resin (catalog number E80215; New England Biolabs) according to the vendor’s instructions for the purification of periplasmic proteins.

    Sequencing:

    Article Title: Importance of the Conserved Carboxyl-Terminal CNOT1 Binding Domain to Tristetraprolin Activity In Vivo
    Article Snippet: Cells were lysed as described above, and supernatants were incubated with amylose resin (New England Biolabs) and eluted with 40 mM maltose. .. Cell-free deadenylation assays were performed as described previously ( , ), using a target mRNA sequence at a final concentration of 200 nM that contained a single TTP family member binding site, 5′-[6FAM/fluorescein]-AAUCAUCC UUAUUUAUU ACCAUUAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA-3′ (the binding site is underlined).

    Article Title: A Tetratricopeptide Repeat Domain Protein has Profound Effects on Assembly of Periplasmic Flagella, Morphology, and Motility of the Lyme disease spirochete Borrelia burgdorferi
    Article Snippet: Subsequently, the codon optimized bb0236 was cloned in pMAL c5x expression vector (NEB Inc.) after removing the signal sequence (a.a. 1–20). .. The soluble fraction was incubated with 150 μl of amylose resin (NEB Inc.) at 4°C for overnight.

    Affinity Chromatography:

    Article Title: Multi-tiered pairing selectivity between E2 ubiquitin–conjugating enzymes and E3 ligases
    Article Snippet: .. The cloning of PUB22 into pMal-c2X vector (New England Biolabs), expression of the MBP-PUB22 in Escherichia coli Rosetta 2(DE3) pLysS and its purification by affinity chromatography using amylose resin (New England Biolabs) was previously described ( ). .. UBA1 and genes encoded the selected E2s ( UBC: 1, 5, 8, 9, 10, 11, 12, 17, 26, 28, 29, 30, 35 ) or MMZ4 were cloned into pENTR/D-TOPO (Invitrogen) entry clone and used for cloning into pDEST17 Gateway vector (Thermo Fisher Scientific) for His-tagged protein expression via LR reaction (Invitrogen).

    Article Title: Cross-cleavage activity of Cas6b in crRNA processing of two different CRISPR-Cas systems in Methanosarcina mazei Gö1
    Article Snippet: .. MBP-fusion proteins were purified from the supernatants by affinity chromatography with amylose resin according to the manufacturer’s instructions (New England BioLabs). .. The purified proteins were dialyzed against 10 mM Tris/HCL pH 7.6, followed by subsequent purification by anion-exchange chromatography using a Q-Seharose FF (XK26, Pharmacia Biotech) as described before [ ].

    Article Title: Dual Role of the C-Terminal Domain in Osmosensing by Bacterial Osmolyte Transporter ProP
    Article Snippet: .. The culture was incubated at 37°C with shaking (200 rotations per minute) for 1–1.5 h. Isopropyl β -D-1-thiogalactopyranoside (1 mM) was added, and the incubation was continued at 25°C for 4 h. The proteins were purified from periplasmic extracts by affinity chromatography using Amylose resin (catalog number E80215; New England Biolabs) according to the vendor’s instructions for the purification of periplasmic proteins. .. The molecular weights of the intact, purified proteins were determined using an Agilent UHD 6530 Q-ToF electrospray mass spectrometer (Agilent, Santa Clara, CA) at the Mass Spectrometry Facility of the Advanced Analysis Centre, University of Guelph.

    Gel Extraction:

    Article Title: Structural Basis for Influenza Virus NS1 Protein Block of mRNA Nuclear Export
    Article Snippet: .. 50xAdvantage Polymerase mix (Clontech (EMD), 639202); dNTP’s (Clontech (EMD), 639125); BamHI-HF (New England Biolabs (NEB), R3136T); NotI (New England Biolabs (NEB), R0189S); SmaI (New England Biolabs (NEB), R0141); XhoI (New England Biolabs (NEB), R0146S); T4 DNA Ligase (New England Biolabs (NEB), M0202); QIAquick Gel Extraction Kit (Qiagen, 28704); NEB® 5-alpha Competent E. coli (New England Biolabs (NEB), ); Rosetta (DE3) Competent Cells (EMD Millipore, 70954); SOC Outgrowth Medium (New England Biolabs (NEB), B9020); QIAprep Spin Miniprep Kit (250) (Qiagen, 27106); Lipofectamine® RNAiMAX Transfection Reagent (Thermo Fisher Scientific, 13778150); RNeasy Plus Mini Kit (Qiagen, 74134); Random Hexamers (50 μM) (Thermo Fisher Scientific, N8080127); Protector RNase Inhibitor (Roche, 03335402001); SuperScript II Reverse Transcriptase (Thermo Fisher Scientific, 18064014); LIGHTCYCLER 480 SYBR GREEN I MASTER (Roche, 04707516001); LightCycler® 480 Multiwell Plate 96, White (Roche, 04729692001); NE-PER™ Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific, 78833); Complete EDTA-free protease inhibitor tablets (Sigma-Aldrich, 11873580001); TransIT-X2® Dynamic Delivery System (Mirus Bio, MIR6000); SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific, 34096); L-Glutathione reduced (Sigma-Aldrich, G4251–25G); Ampicillin (Sigma-Aldrich, A-9518); Kanamycin Monosulfate (Gold Biotechnology, K-120–10); IPTG (Gold Biotechnology, I2481C50); Imidazole (Sigma-Aldrich, 56750); PMSF (RPI, ); Aprotinin (Santa Cruz Biotechnology, sc-3595); Leupeptin (Santa Cruz Biotechnology, sc295358); Pepstatin A (Thermo Fisher Scientific, ); Glutathione Sepharose 4B (GE Healthcare, 17–0756-01); Amylose Resin (New England Biolabs (NEB), E8021S); Ni-NTA Agarose (Qiagen, 30210); Mono Q 5/50 GL (GE Healthcare, 17-5166-01); HiTrap SP HP (GE Healthcare, 17-1151-01); Superdex 200 HR 10/30 (GE Healthcare, 17-1088-01); EasyTag EXPRESS35 S Protein Labeling Mix, [35S]-, 7mCi (PerkinElmer, NEG772007MC); T7 RiboMAX™ Express Large Scale RNA Production System (Promega, P1320); Protein G Sepharose® 4 Fast Flow (GE Healthcare, 17-0618-01); Hoechst 33258 (Molecular Probes/Life Technologies); M mRNA probes (Biosearch Technologies) ; ProLong Gold antifade reagent (Life Technologies, ). ..

    SDS Page:

    Article Title: NRDE2 negatively regulates exosome functions by inhibiting MTR4 recruitment and exosome interaction
    Article Snippet: For each pull-down reaction, 8 μg of MBP-tagged proteins and 8 μg of His-tagged proteins were added to 20 μL of Amylose Resin (New England BioLabs) in pull-down buffer (1× PBS, 0.1% Triton, 0.2 mM PMSF, protease inhibitor). .. The samples were separated by SDS-PAGE followed by Coomassie staining.

    Article Title: Protein interaction mapping identifies RBBP6 as a negative regulator of Ebola virus replication
    Article Snippet: Amylose resin (NEB) was equilibrated with buffer containing 20 mM Tris pH 7.5, 150 mM NaCl, and 5 mM β-ME. .. Gel samples were taken at each step and visualized on SDS-PAGE by Coomassie blue staining.

    Plasmid Preparation:

    Article Title: The nuclear transcription factor Rtg1p functions as a cytosolic, post-transcriptional regulator in the methylotrophic yeast Pichia pastoris
    Article Snippet: The PCR product was digested with BamHI and PstI and cloned into pMAL-c2X vector. .. Recombinant proteins were expressed as MBP fusion proteins (PpRtgXp, PpRtgXp*, and ScRtg3p) or GST fusion proteins (PpRTG1 and ScRTG1) and purified using amylose resin (New England Biolabs) and GSH-agarose beads (G Biosciences), respectively, according to the manufacturers' instructions.

    Article Title: Multi-tiered pairing selectivity between E2 ubiquitin–conjugating enzymes and E3 ligases
    Article Snippet: .. The cloning of PUB22 into pMal-c2X vector (New England Biolabs), expression of the MBP-PUB22 in Escherichia coli Rosetta 2(DE3) pLysS and its purification by affinity chromatography using amylose resin (New England Biolabs) was previously described ( ). .. UBA1 and genes encoded the selected E2s ( UBC: 1, 5, 8, 9, 10, 11, 12, 17, 26, 28, 29, 30, 35 ) or MMZ4 were cloned into pENTR/D-TOPO (Invitrogen) entry clone and used for cloning into pDEST17 Gateway vector (Thermo Fisher Scientific) for His-tagged protein expression via LR reaction (Invitrogen).

    Article Title: Importance of the Conserved Carboxyl-Terminal CNOT1 Binding Domain to Tristetraprolin Activity In Vivo
    Article Snippet: The human TTP TZF domain fusion peptide was expressed in BL21(DE3) cells (Millipore-Sigma) after induction with 0.1 mM IPTG for 16 h at 20°C and was purified as described above for full-length and mutant TTP proteins, with the exception that the affinity tag was removed following elution from the HisTrap column by incubation with tobacco etch virus (TEV) protease (vector kindly supplied by Traci T. Hall, NIEHS) for 16 h at 4°C in the presence of 2 mM DTT. .. Cells were lysed as described above, and supernatants were incubated with amylose resin (New England Biolabs) and eluted with 40 mM maltose.

    Article Title: A Tetratricopeptide Repeat Domain Protein has Profound Effects on Assembly of Periplasmic Flagella, Morphology, and Motility of the Lyme disease spirochete Borrelia burgdorferi
    Article Snippet: E. coli pLysS cells harboring plasmid BB0236-pMAL c5x or the empty vector pMAL c5x were induced with 1 mM of IPTG for four hours followed by harvesting the cells. .. The soluble fraction was incubated with 150 μl of amylose resin (NEB Inc.) at 4°C for overnight.

    Article Title: Dual Role of the C-Terminal Domain in Osmosensing by Bacterial Osmolyte Transporter ProP
    Article Snippet: Proteins MalE∗ and MalE∗ ProP Xc CTD were purified from transformants of E. coli strain DH5 α ( ) containing plasmid pDC369 and pDC361, respectively. .. The culture was incubated at 37°C with shaking (200 rotations per minute) for 1–1.5 h. Isopropyl β -D-1-thiogalactopyranoside (1 mM) was added, and the incubation was continued at 25°C for 4 h. The proteins were purified from periplasmic extracts by affinity chromatography using Amylose resin (catalog number E80215; New England Biolabs) according to the vendor’s instructions for the purification of periplasmic proteins.

    Article Title: A Hippo-like signalling pathway controls tracheal morphogenesis in Drosophila melanogaster
    Article Snippet: The C-terminal hydrophobic fragments of D. melanogaster Trc (residues 308 to 459), human NDR1 (residues 301 to 465) and NDR2 (residues 302 to 464) were inserted into the pMal-c2 vector using BamH I and Xho I/ Sal I sites to generate pMal-Trc/NDR(HM) plasmids. .. Recombinant Mal-fusion proteins were expressed in E. coli BL21(DE3) at 30°C and purified using amylose resin (E8021, New England BioLabs) as described ( ).

    Software:

    Article Title: Dual Role of the C-Terminal Domain in Osmosensing by Bacterial Osmolyte Transporter ProP
    Article Snippet: The culture was incubated at 37°C with shaking (200 rotations per minute) for 1–1.5 h. Isopropyl β -D-1-thiogalactopyranoside (1 mM) was added, and the incubation was continued at 25°C for 4 h. The proteins were purified from periplasmic extracts by affinity chromatography using Amylose resin (catalog number E80215; New England Biolabs) according to the vendor’s instructions for the purification of periplasmic proteins. .. Data analysis was performed using the MassHunter Qualitative Analysis version B.08.00 (Agilent) software.

    Binding Assay:

    Article Title: Importance of the Conserved Carboxyl-Terminal CNOT1 Binding Domain to Tristetraprolin Activity In Vivo
    Article Snippet: Cells were lysed as described above, and supernatants were incubated with amylose resin (New England Biolabs) and eluted with 40 mM maltose. .. Cell-free deadenylation assays were performed as described previously ( , ), using a target mRNA sequence at a final concentration of 200 nM that contained a single TTP family member binding site, 5′-[6FAM/fluorescein]-AAUCAUCC UUAUUUAUU ACCAUUAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA-3′ (the binding site is underlined).

    Article Title: The Polycomb-Group Repressor MEDEA Attenuates Pathogen Defense
    Article Snippet: MBP and MBP-Di19 were expressed in the Escherichia coli BL21 (DE3) strain and purified using amylose resin (New England Biolabs). .. For the binding assay, 400 ng, 1 µg, 1.6 µg and 2 µg of MBP or MBP Di19 was used.

    Article Title: Dual Role of the C-Terminal Domain in Osmosensing by Bacterial Osmolyte Transporter ProP
    Article Snippet: Paragraph title: Protein purification and liposome binding assays ... The culture was incubated at 37°C with shaking (200 rotations per minute) for 1–1.5 h. Isopropyl β -D-1-thiogalactopyranoside (1 mM) was added, and the incubation was continued at 25°C for 4 h. The proteins were purified from periplasmic extracts by affinity chromatography using Amylose resin (catalog number E80215; New England Biolabs) according to the vendor’s instructions for the purification of periplasmic proteins.

    Article Title: A Hippo-like signalling pathway controls tracheal morphogenesis in Drosophila melanogaster
    Article Snippet: Full-length D. melanogaster GckIII and human MST3, MST4 or STK25 cDNAs were inserted into the pMal-c2 vector (New England BioLabs) using BamH I and Xho I/ Sal I or Xba I sites to generate pMal-GckIII/GCKIII plasmids, which can express kinase-dead versions that are N-terminally tagged by the maltose binding protein (Mal). .. Recombinant Mal-fusion proteins were expressed in E. coli BL21(DE3) at 30°C and purified using amylose resin (E8021, New England BioLabs) as described ( ).

    In Vitro:

    Article Title: Importance of the Conserved Carboxyl-Terminal CNOT1 Binding Domain to Tristetraprolin Activity In Vivo
    Article Snippet: Paragraph title: In vitro deadenylation assays. ... Cells were lysed as described above, and supernatants were incubated with amylose resin (New England Biolabs) and eluted with 40 mM maltose.

    Article Title: Inheritance of CENP-A nucleosomes during DNA replication requires HJURP
    Article Snippet: Paragraph title: In vitro recombinant protein pull-downs ... Thirty microliters of 50 μM MBP-His-MCM243−160 was incubated with 20 μl of amylose resin (New England Biolabs) in P300 buffer (NaP pH 7.0, 300mM NaCl).

    Article Title: Protein interaction mapping identifies RBBP6 as a negative regulator of Ebola virus replication
    Article Snippet: Paragraph title: In vitro Pull-down Assays ... Amylose resin (NEB) was equilibrated with buffer containing 20 mM Tris pH 7.5, 150 mM NaCl, and 5 mM β-ME.

    Size-exclusion Chromatography:

    Article Title: A bifunctional role for the UHRF1 UBL domain in the control of hemi-methylated DNA-dependent histone ubiquitylation
    Article Snippet: Alternatively, the His6 -MBP tag was removed with a combination of recapturing the tag on Ni-NTA resin after dialysis into 25 mM Tris-HCl pH 7.6, 200 mM NaCl, and 5 mM imidazole, and passage through amylose resin (New England BioLabs, cat# E8021S). .. Full-length UHRF1 and mutants, DUBL, and all proteins used for NMR experiments were further purified by size exclusion chromatography (SEC) with a Superdex 200 HiLoad 16/600 column (GE Healthcare, Cat #28989335), a Superdex 200 10/300 GL column (GE Healthcare Cat #17517501), or a Superdex 75 10/300 GL column (GE Healthcare, Cat# 17517401).

    Homogenization:

    Article Title: A bifunctional role for the UHRF1 UBL domain in the control of hemi-methylated DNA-dependent histone ubiquitylation
    Article Snippet: Phenylmethylsulfonyl fluoride (PMSF; Sigma, Cat# 329–98-6) was added to a final concentration 1 mM to the resuspended cell pellet immediately prior to cell lyses, which was performed using a short sonication, followed by homogenization or by two passages through a French pressure cell. .. Alternatively, the His6 -MBP tag was removed with a combination of recapturing the tag on Ni-NTA resin after dialysis into 25 mM Tris-HCl pH 7.6, 200 mM NaCl, and 5 mM imidazole, and passage through amylose resin (New England BioLabs, cat# E8021S).

    Nuclear Magnetic Resonance:

    Article Title: A bifunctional role for the UHRF1 UBL domain in the control of hemi-methylated DNA-dependent histone ubiquitylation
    Article Snippet: Alternatively, the His6 -MBP tag was removed with a combination of recapturing the tag on Ni-NTA resin after dialysis into 25 mM Tris-HCl pH 7.6, 200 mM NaCl, and 5 mM imidazole, and passage through amylose resin (New England BioLabs, cat# E8021S). .. Full-length UHRF1 and mutants, DUBL, and all proteins used for NMR experiments were further purified by size exclusion chromatography (SEC) with a Superdex 200 HiLoad 16/600 column (GE Healthcare, Cat #28989335), a Superdex 200 10/300 GL column (GE Healthcare Cat #17517501), or a Superdex 75 10/300 GL column (GE Healthcare, Cat# 17517401).

    Immunoprecipitation:

    Article Title: A Hippo-like signalling pathway controls tracheal morphogenesis in Drosophila melanogaster
    Article Snippet: Recombinant Mal-fusion proteins were expressed in E. coli BL21(DE3) at 30°C and purified using amylose resin (E8021, New England BioLabs) as described ( ). .. To produce immunopurified full-length HA-tagged Tao-1 or TAO1 kinase version, D. melanogaster S2R+ or human HEK293 cells were transfected with pAW_HA-Tao1 or pcDNA3_HA-TAO1 and processed for immunoprecipitation (IP) using anti-HA antibody and stringent IP conditions as described ( ).

    Staining:

    Article Title: NRDE2 negatively regulates exosome functions by inhibiting MTR4 recruitment and exosome interaction
    Article Snippet: For each pull-down reaction, 8 μg of MBP-tagged proteins and 8 μg of His-tagged proteins were added to 20 μL of Amylose Resin (New England BioLabs) in pull-down buffer (1× PBS, 0.1% Triton, 0.2 mM PMSF, protease inhibitor). .. The samples were separated by SDS-PAGE followed by Coomassie staining.

    Article Title: The nuclear transcription factor Rtg1p functions as a cytosolic, post-transcriptional regulator in the methylotrophic yeast Pichia pastoris
    Article Snippet: Recombinant proteins were expressed as MBP fusion proteins (PpRtgXp, PpRtgXp*, and ScRtg3p) or GST fusion proteins (PpRTG1 and ScRTG1) and purified using amylose resin (New England Biolabs) and GSH-agarose beads (G Biosciences), respectively, according to the manufacturers' instructions. .. GST-Rtg1p bound to GSH-agarose beads was incubated with E. coli cell lysates containing MBP-Rtg3p, MBP-RtgXp, or MBP-RtgXp* for 1 h. After centrifugation and washing twice with buffer A, proteins bound to GSH beads were resolved on an SDS-polyacrylamide gel and visualized by Coomassie Brilliant Blue R staining.

    Article Title: Protein interaction mapping identifies RBBP6 as a negative regulator of Ebola virus replication
    Article Snippet: Amylose resin (NEB) was equilibrated with buffer containing 20 mM Tris pH 7.5, 150 mM NaCl, and 5 mM β-ME. .. Gel samples were taken at each step and visualized on SDS-PAGE by Coomassie blue staining.

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  • 90
    New England Biolabs amylose resin
    Amylose Resin, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 90/100, based on 736 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/amylose resin/product/New England Biolabs
    Average 90 stars, based on 736 article reviews
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    amylose resin - by Bioz Stars, 2020-01
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