tris hcl  (Millipore)

 
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  • 86
    Name:
    Tris HCl Buffer
    Description:

    Catalog Number:
    t6455
    Price:
    None
    Applications:
    Tris-HCl Buffer, pH 10, 10x, Antigen Retriever has been used as a heat-induced antigen retriever on formalin-fixed paraffin-embedded (FFPE) tissue sections prior to application of antibodies. In immunohistochemistry (IHC), most commonly used fixatives such as formalin mask tissue antigens (cellular, membrane, and nuclear) by their intrinsic crosslinking. This masking results in poor or no staining in IHC. The use of Tris-HCl buffer, pH 10, or other antigen retrieval solutions on FFPE tissue sections improves accessibility of antibodies to tissue antigens.
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    Structured Review

    Millipore tris hcl
    Purification and characterized of Influenza D nucleoprotein. ( a ) Size exclusion chromatography profile of wild-type D/NP. The sample was loaded on a Hiload TM 16/600 S200 column equilibrated with the running buffer 20 mM <t>Tris-HCl</t> pH 7.5, 300 mM <t>NaCl</t> and 5 mM β-mercaptoethanol. ( b ) SEC-MALLS-RI analysis of D/NP. SEC was performed with a Superdex TM 200 increase 10/300 GL column equilibrated with 20 mM Tris-HCl pH 7.5, 150 mM NaCl and 5 mM β-ME. The panel shows the theoretical Mw and the measured Mw. ( c ) and ( e ) Electron microscopy images of the elution peak of D/NP and D/NP-511. Samples show different oligomeric states although most oligomers are tetramers. The scale bar corresponds to 100 nm. ( d ) Coomassie blue-stained SDS-PAGE (4–20% gradient polyacrylamide) showing the purified wild-type D/NP and the two C-terminal truncated mutants (D/NP-529 and D/NP-511).

    https://www.bioz.com/result/tris hcl/product/Millipore
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    tris hcl - by Bioz Stars, 2021-03
    86/100 stars

    Images

    1) Product Images from "The structure of the nucleoprotein of Influenza D shows that all Orthomyxoviridae nucleoproteins have a similar NPCORE, with or without a NPTAIL for nuclear transport"

    Article Title: The structure of the nucleoprotein of Influenza D shows that all Orthomyxoviridae nucleoproteins have a similar NPCORE, with or without a NPTAIL for nuclear transport

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-37306-y

    Purification and characterized of Influenza D nucleoprotein. ( a ) Size exclusion chromatography profile of wild-type D/NP. The sample was loaded on a Hiload TM 16/600 S200 column equilibrated with the running buffer 20 mM Tris-HCl pH 7.5, 300 mM NaCl and 5 mM β-mercaptoethanol. ( b ) SEC-MALLS-RI analysis of D/NP. SEC was performed with a Superdex TM 200 increase 10/300 GL column equilibrated with 20 mM Tris-HCl pH 7.5, 150 mM NaCl and 5 mM β-ME. The panel shows the theoretical Mw and the measured Mw. ( c ) and ( e ) Electron microscopy images of the elution peak of D/NP and D/NP-511. Samples show different oligomeric states although most oligomers are tetramers. The scale bar corresponds to 100 nm. ( d ) Coomassie blue-stained SDS-PAGE (4–20% gradient polyacrylamide) showing the purified wild-type D/NP and the two C-terminal truncated mutants (D/NP-529 and D/NP-511).
    Figure Legend Snippet: Purification and characterized of Influenza D nucleoprotein. ( a ) Size exclusion chromatography profile of wild-type D/NP. The sample was loaded on a Hiload TM 16/600 S200 column equilibrated with the running buffer 20 mM Tris-HCl pH 7.5, 300 mM NaCl and 5 mM β-mercaptoethanol. ( b ) SEC-MALLS-RI analysis of D/NP. SEC was performed with a Superdex TM 200 increase 10/300 GL column equilibrated with 20 mM Tris-HCl pH 7.5, 150 mM NaCl and 5 mM β-ME. The panel shows the theoretical Mw and the measured Mw. ( c ) and ( e ) Electron microscopy images of the elution peak of D/NP and D/NP-511. Samples show different oligomeric states although most oligomers are tetramers. The scale bar corresponds to 100 nm. ( d ) Coomassie blue-stained SDS-PAGE (4–20% gradient polyacrylamide) showing the purified wild-type D/NP and the two C-terminal truncated mutants (D/NP-529 and D/NP-511).

    Techniques Used: Purification, Size-exclusion Chromatography, Electron Microscopy, Staining, SDS Page

    Interaction of D/NP and D/NP TAIL with importin-α7. ( a ) Size exclusion chromatography profile of a mixture between human importin-α7 and D/NP TAIL . The mixture (molar ratio 1 importin-α7 for 2 D/NP TAIL ) was incubated 1 hour at room temperature and then loaded on a Superdex TM 75 10/300GL column equilibrated with the running buffer 20 mM Tris-HCl pH 7.5, 250 mM NaCl, 5 mM β-mercaptoethanol. ( b ) Thermal stability assay of importin-α7 in absence (green) or in presence (red) of D/NP TAIL . In presence of D/NP TAIL , the melting temperature of importin-α7 is 5 °C higher. D/NP TAIL alone using Thermofluor did not give any denaturation signal (yellow curve). The upper insert corresponds to the derivative of the fluorescence signal for a precise measure of the melting temperature. ( c ) Affinity of importin-α7 for D/NP TAIL by measured by surface plasmon resonance (SPR). Biotinylated D/NP TAIL (left) and control peptide (right) were captured on a streptavidin-coated sensor chip surface before injections of several importin-α7 concentrations (10 nM in red, 25 nM in orange, 50 nM in green, 75 nM in blue and 100 nM in purple). The sensorgrams of the interaction between D/NP TAIL and importin-α7 were fitted under a Langmuir 1:1 binding model with mass-transfer (black line). ( d ) SEC-MALLS analysis of D/NP in complex with importin-α7. The mixture (molar ratio 1 D/NP for 1.2 importin-α7) was incubated 1 hour at room temperature and then loaded on a Superdex TM 200 increase 10/300 GL. The experimental molecular weight is consistent with the expected mass of four importins-α7 bound per D/NP tetramer. ( e ) Pull-down assays of human importin-α7 by D/NP and the two C-terminal truncated mutants (D/NP-529 and D/NP-511). The his-tags are on D/NP. The mixtures (molar ratio 1 D/NP for 1.2 importin-α7) were incubated 1 hour and the experience was done as described in panel ( a ). The figure shows the coomassie blue-stained SDS-PAGE (12% polyacrylamide) with the Load, FlowThough, Wash and the second fractions (E2).
    Figure Legend Snippet: Interaction of D/NP and D/NP TAIL with importin-α7. ( a ) Size exclusion chromatography profile of a mixture between human importin-α7 and D/NP TAIL . The mixture (molar ratio 1 importin-α7 for 2 D/NP TAIL ) was incubated 1 hour at room temperature and then loaded on a Superdex TM 75 10/300GL column equilibrated with the running buffer 20 mM Tris-HCl pH 7.5, 250 mM NaCl, 5 mM β-mercaptoethanol. ( b ) Thermal stability assay of importin-α7 in absence (green) or in presence (red) of D/NP TAIL . In presence of D/NP TAIL , the melting temperature of importin-α7 is 5 °C higher. D/NP TAIL alone using Thermofluor did not give any denaturation signal (yellow curve). The upper insert corresponds to the derivative of the fluorescence signal for a precise measure of the melting temperature. ( c ) Affinity of importin-α7 for D/NP TAIL by measured by surface plasmon resonance (SPR). Biotinylated D/NP TAIL (left) and control peptide (right) were captured on a streptavidin-coated sensor chip surface before injections of several importin-α7 concentrations (10 nM in red, 25 nM in orange, 50 nM in green, 75 nM in blue and 100 nM in purple). The sensorgrams of the interaction between D/NP TAIL and importin-α7 were fitted under a Langmuir 1:1 binding model with mass-transfer (black line). ( d ) SEC-MALLS analysis of D/NP in complex with importin-α7. The mixture (molar ratio 1 D/NP for 1.2 importin-α7) was incubated 1 hour at room temperature and then loaded on a Superdex TM 200 increase 10/300 GL. The experimental molecular weight is consistent with the expected mass of four importins-α7 bound per D/NP tetramer. ( e ) Pull-down assays of human importin-α7 by D/NP and the two C-terminal truncated mutants (D/NP-529 and D/NP-511). The his-tags are on D/NP. The mixtures (molar ratio 1 D/NP for 1.2 importin-α7) were incubated 1 hour and the experience was done as described in panel ( a ). The figure shows the coomassie blue-stained SDS-PAGE (12% polyacrylamide) with the Load, FlowThough, Wash and the second fractions (E2).

    Techniques Used: Size-exclusion Chromatography, Incubation, Stability Assay, Fluorescence, SPR Assay, Chromatin Immunoprecipitation, Binding Assay, Molecular Weight, Staining, SDS Page

    Related Articles

    Purification:

    Article Title: Using singular perturbation theory to determine kinetic parameters in a non-standard coupled enzyme assay
    Article Snippet: We then incubated this culture at 22 \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$^{\circ }$$\end{document} ∘ C for another 10 h and harvested it by centrifugation at \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$2800{\times }g$$\end{document} 2800 × g , at 4 \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$^{\circ }$$\end{document} ∘ C. .. Purification of chitin-tagged CnAptA transaminase We re-suspended the cell pellets from the previous centrifugation step in Tris-HCl buffer (20mM Tris-HCl, 0.5 M NaCl [pH 7.4]) and disrupted them using a Soniprep 150 MSE SANYO sonicator, at 4 \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$^{\circ }$$\end{document} ∘ C, at 30 s intervals for 30 min. We removed the insoluble fraction by centrifugation at \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$8400 \times g$$\end{document} 8400 × g for 30 min. Then, we loaded the supernatant onto the chromatography column packed with the chitin resin (Poly-Prep® prepacked columns, AG® 1-X8, chloride form #731-1550), equilibrated with the same buffer at pH 8.4, according to the suppliers protocol. .. We induced the on-column cleavage of the CnAptA purified enzyme by adding Tris-HCl buffer containing 1,4-Dithiothreitol (DDT) (20 mM Tris-HCl, 0.5 M NaCl [pH 8.4]), 50 mM DTT).

    Centrifugation:

    Article Title: Using singular perturbation theory to determine kinetic parameters in a non-standard coupled enzyme assay
    Article Snippet: We then incubated this culture at 22 \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$^{\circ }$$\end{document} ∘ C for another 10 h and harvested it by centrifugation at \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$2800{\times }g$$\end{document} 2800 × g , at 4 \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$^{\circ }$$\end{document} ∘ C. .. Purification of chitin-tagged CnAptA transaminase We re-suspended the cell pellets from the previous centrifugation step in Tris-HCl buffer (20mM Tris-HCl, 0.5 M NaCl [pH 7.4]) and disrupted them using a Soniprep 150 MSE SANYO sonicator, at 4 \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$^{\circ }$$\end{document} ∘ C, at 30 s intervals for 30 min. We removed the insoluble fraction by centrifugation at \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$8400 \times g$$\end{document} 8400 × g for 30 min. Then, we loaded the supernatant onto the chromatography column packed with the chitin resin (Poly-Prep® prepacked columns, AG® 1-X8, chloride form #731-1550), equilibrated with the same buffer at pH 8.4, according to the suppliers protocol. .. We induced the on-column cleavage of the CnAptA purified enzyme by adding Tris-HCl buffer containing 1,4-Dithiothreitol (DDT) (20 mM Tris-HCl, 0.5 M NaCl [pH 8.4]), 50 mM DTT).

    Article Title: Physiological Effect of XoxG(4) on Lanthanide-Dependent Methanotrophy
    Article Snippet: .. The samples were then desalted, and the buffer was replaced by dilution with 100 mM Tris-HCl (pH 9.0) buffer (20-fold, repeated five times), followed by concentration by centrifugation in 10-kDa-cutoff dialysis tubes (Millipore, Billerica, MA), in the case of XoxG and in 50-kDa cutoff tubes in the case of XoxF until the calculated concentration of imidazole reached less than 1 μM. .. The purified proteins were analyzed by separation in a 12% SDS-denaturing polyacrylamide gel, followed by Coomassie blue staining.

    Chromatography:

    Article Title: Using singular perturbation theory to determine kinetic parameters in a non-standard coupled enzyme assay
    Article Snippet: We then incubated this culture at 22 \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$^{\circ }$$\end{document} ∘ C for another 10 h and harvested it by centrifugation at \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$2800{\times }g$$\end{document} 2800 × g , at 4 \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$^{\circ }$$\end{document} ∘ C. .. Purification of chitin-tagged CnAptA transaminase We re-suspended the cell pellets from the previous centrifugation step in Tris-HCl buffer (20mM Tris-HCl, 0.5 M NaCl [pH 7.4]) and disrupted them using a Soniprep 150 MSE SANYO sonicator, at 4 \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$^{\circ }$$\end{document} ∘ C, at 30 s intervals for 30 min. We removed the insoluble fraction by centrifugation at \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$8400 \times g$$\end{document} 8400 × g for 30 min. Then, we loaded the supernatant onto the chromatography column packed with the chitin resin (Poly-Prep® prepacked columns, AG® 1-X8, chloride form #731-1550), equilibrated with the same buffer at pH 8.4, according to the suppliers protocol. .. We induced the on-column cleavage of the CnAptA purified enzyme by adding Tris-HCl buffer containing 1,4-Dithiothreitol (DDT) (20 mM Tris-HCl, 0.5 M NaCl [pH 8.4]), 50 mM DTT).

    other:

    Article Title: Mycobacterium tuberculosis Rv3194c efficiently facilitates mycobacterial-lung epithelial interaction through its HA-binding site
    Article Snippet: The desired protein was then eluted by 200 mM NaCl in Tris-HCl buffer at pH 7.4.

    Article Title: Substitution of nucleotide-sugar by trehalose-dependent glycogen synthesis pathways in Chlamydiales underlines an unusual requirement for storage polysaccharides within obligate intracellular bacteria
    Article Snippet: Kinetic parameters of GlgE-EL were determined in triplicates at 30°C in 15 mM Tris/HCl buffer at pH 6.8.

    Lysis:

    Article Title: New aspects of antiproliferative activity of 4-hydroxybenzyl isothiocyanate, a natural H2S-donor
    Article Snippet: .. On the following day, the cells were treated with various concentrations of HBITC or an equal volume of DMSO in the culture medium for 24 and 48 h. Proteins from the cells were extracted in lysis buffer containing 50 mM Tris–HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.5% NP-40, and 1X Complete Protease Inhibitor Cocktail (Sigma-Aldrich Corp., St. Louis, MO, USA). ..

    Protease Inhibitor:

    Article Title: New aspects of antiproliferative activity of 4-hydroxybenzyl isothiocyanate, a natural H2S-donor
    Article Snippet: .. On the following day, the cells were treated with various concentrations of HBITC or an equal volume of DMSO in the culture medium for 24 and 48 h. Proteins from the cells were extracted in lysis buffer containing 50 mM Tris–HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.5% NP-40, and 1X Complete Protease Inhibitor Cocktail (Sigma-Aldrich Corp., St. Louis, MO, USA). ..

    Concentration Assay:

    Article Title: Physiological Effect of XoxG(4) on Lanthanide-Dependent Methanotrophy
    Article Snippet: .. The samples were then desalted, and the buffer was replaced by dilution with 100 mM Tris-HCl (pH 9.0) buffer (20-fold, repeated five times), followed by concentration by centrifugation in 10-kDa-cutoff dialysis tubes (Millipore, Billerica, MA), in the case of XoxG and in 50-kDa cutoff tubes in the case of XoxF until the calculated concentration of imidazole reached less than 1 μM. .. The purified proteins were analyzed by separation in a 12% SDS-denaturing polyacrylamide gel, followed by Coomassie blue staining.

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  • 95
    Millipore trizma hydrochloride tris hcl
    The reusability of the DNAzyme/GOx/PS nanofibrous membrane by separately measuring its absorbance in five cycles of colorimetric system and <t>Tris</t> buffer, respectively. The concentration of target HIV is 100 nM.
    Trizma Hydrochloride Tris Hcl, supplied by Millipore, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/trizma hydrochloride tris hcl/product/Millipore
    Average 95 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    trizma hydrochloride tris hcl - by Bioz Stars, 2021-03
    95/100 stars
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    97
    Millipore tris hcl
    The effect of pH on the degradation of protein substrates Hb, azocasein, fibrinogen and goat IgG by recombinant <t>HC58</t> protein of adult H. contortus . The buffers 0.1 M acetate, 0.1 M phosphate, 0.1 M <t>Tris</t> and 0.1 M glycine with overlapping pH, in the ranges pH 3 to 11, supplemented with antibiotics were used.
    Tris Hcl, supplied by Millipore, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/tris hcl/product/Millipore
    Average 97 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    tris hcl - by Bioz Stars, 2021-03
    97/100 stars
      Buy from Supplier


    Image Search Results


    The reusability of the DNAzyme/GOx/PS nanofibrous membrane by separately measuring its absorbance in five cycles of colorimetric system and Tris buffer, respectively. The concentration of target HIV is 100 nM.

    Journal: Scientific Reports

    Article Title: Ultrasensitive Visual Detection of HIV DNA Biomarkers via a Multi-amplification Nanoplatform

    doi: 10.1038/srep23949

    Figure Lengend Snippet: The reusability of the DNAzyme/GOx/PS nanofibrous membrane by separately measuring its absorbance in five cycles of colorimetric system and Tris buffer, respectively. The concentration of target HIV is 100 nM.

    Article Snippet: Materials and Reagents The polystyrene (PS, M w ~ 280000), N, N-dimethylformamide (DMF), GOx (153 U/mg), avidin (12.8 U/mg), hemin, dimethyl sulfoxide (DMSO), ABTS, Stains-All, and trizma® hydrochloride (Tris-HCl) were purchased from Sigma-Aldrich (St. Louis, MO, USA).

    Techniques: Concentration Assay

    The effect of pH on the degradation of protein substrates Hb, azocasein, fibrinogen and goat IgG by recombinant HC58 protein of adult H. contortus . The buffers 0.1 M acetate, 0.1 M phosphate, 0.1 M Tris and 0.1 M glycine with overlapping pH, in the ranges pH 3 to 11, supplemented with antibiotics were used.

    Journal: Journal of Veterinary Science

    Article Title: Characterization of HC58cDNA, a putative cysteine protease from the parasite Haemonchus contortus

    doi: 10.4142/jvs.2006.7.3.249

    Figure Lengend Snippet: The effect of pH on the degradation of protein substrates Hb, azocasein, fibrinogen and goat IgG by recombinant HC58 protein of adult H. contortus . The buffers 0.1 M acetate, 0.1 M phosphate, 0.1 M Tris and 0.1 M glycine with overlapping pH, in the ranges pH 3 to 11, supplemented with antibiotics were used.

    Article Snippet: The recombinant HC58 protein was refolded in 0.1 M urea, 50 mM Tris-HCl, 2 M oxidized glutathione (Sigma, USA), and 0.02 M reduced glutathione (pH 8.0; Sigma, USA).

    Techniques: Recombinant

    Mass spectrometry analysis after in -solution digestion of the protein BmrA. The purified protein BmrA (10 µM) was digested using trypsin in the solution containing 0.05% DDM, 50 mM NaCl, 50 mM Tris/HCl pH 8, 10% glycerol and 5 mM beta-mercaptoethanol. The mixture was investigated by mass spectrometry in ESI or APPI under dopant assisted conditions using toluene and 9 eV photons. The precursor ion m/z 1198.75 corresponding to the first transmembrane domain is labelled with a black arrow.

    Journal: PLoS ONE

    Article Title: Characterization of Hydrophobic Peptides in the Presence of Detergent by Photoionization Mass Spectrometry

    doi: 10.1371/journal.pone.0079033

    Figure Lengend Snippet: Mass spectrometry analysis after in -solution digestion of the protein BmrA. The purified protein BmrA (10 µM) was digested using trypsin in the solution containing 0.05% DDM, 50 mM NaCl, 50 mM Tris/HCl pH 8, 10% glycerol and 5 mM beta-mercaptoethanol. The mixture was investigated by mass spectrometry in ESI or APPI under dopant assisted conditions using toluene and 9 eV photons. The precursor ion m/z 1198.75 corresponding to the first transmembrane domain is labelled with a black arrow.

    Article Snippet: BmrA at concentration of 10 µM in 200 µL containing 50 mM Tris/HCl, pH 8, 50 mM NaCl, 10% glycerol, 0.05% DDM, and 5 mM β-mercaptoethanol was reduced with 10 mM DTT (Sigma, Saint-Quentin Fallavier, France) at 56°C for 45 minutes followed by alkylation with 40 mM iodoacetamide (Sigma) at room temperature in the dark for 1 hour.

    Techniques: Mass Spectrometry, Purification

    DNase I Footprinting DNase I footprint of the WT and KRAA UvrA proteins bound to the 50 bp F 26 50/NBD duplex with the 32 P on the 5′ end of the damaged strand. The indicated proteins were incubated with 2 nM duplex in the presence of 50 mM Tris-HCl, pH 7.5, 50 mM KCl, 10 MgCl 2 , 1 mM ATP, 10 mM DTT, 100 nM bovine serum albumin for 15 min at 37 °C then DNase I treated and processed as described in the methods. Panel a , WT UvrA DNase I footprint. Panel b , KRAA UvrA DNase I footprint. Both gels are loaded in the same order. The lanes contained the following: lane 1, no protein and no DNase I; lane 2, UvrA 2 , 10 nM and no DNase I; lane 3, no protein plus DNase I; lanes 4–7, increasing concentrations of UvrA 2 , 10 – 80 nM, plus DNase I; lane 8, UvrA 2 , 10 nM, WT UvrB, 100 nM, plus DNase I; lane 9, WT UvrB, 100 nM, plus DNase I. The position of the adduct and the 3′ and 5′ incision sites are noted on the left-hand side of the gels. The bands that were quantified for the graph in panel c are denoted by the arrows, p1 and p2. Panel c ]. For more details see the Materials and Methods.

    Journal: DNA repair

    Article Title: Cooperative damage recognition by UvrA and UvrB: Identification of UvrA residues that mediate DNA binding

    doi: 10.1016/j.dnarep.2007.11.013

    Figure Lengend Snippet: DNase I Footprinting DNase I footprint of the WT and KRAA UvrA proteins bound to the 50 bp F 26 50/NBD duplex with the 32 P on the 5′ end of the damaged strand. The indicated proteins were incubated with 2 nM duplex in the presence of 50 mM Tris-HCl, pH 7.5, 50 mM KCl, 10 MgCl 2 , 1 mM ATP, 10 mM DTT, 100 nM bovine serum albumin for 15 min at 37 °C then DNase I treated and processed as described in the methods. Panel a , WT UvrA DNase I footprint. Panel b , KRAA UvrA DNase I footprint. Both gels are loaded in the same order. The lanes contained the following: lane 1, no protein and no DNase I; lane 2, UvrA 2 , 10 nM and no DNase I; lane 3, no protein plus DNase I; lanes 4–7, increasing concentrations of UvrA 2 , 10 – 80 nM, plus DNase I; lane 8, UvrA 2 , 10 nM, WT UvrB, 100 nM, plus DNase I; lane 9, WT UvrB, 100 nM, plus DNase I. The position of the adduct and the 3′ and 5′ incision sites are noted on the left-hand side of the gels. The bands that were quantified for the graph in panel c are denoted by the arrows, p1 and p2. Panel c ]. For more details see the Materials and Methods.

    Article Snippet: ATP (Roche) was added to a final concentration of 1 mM in a 100 µl reaction mixture containing 50 mM Tris-HCl pH 7.5, 55 mM KCl, 4 mM MgCl2 , 1 mM DTT, 12.6 units/ml L-lactic dehydrogenase (Sigma), 10 units/ml pyruvate kinase (Sigma), 2 mM phosphoenol pyruvate (Roche), 0.15 mM NADH (Roche), 50 nM Bca UvrA (WT or mutants) and 100 nM Bca UvrB.

    Techniques: Footprinting, Incubation