dithiothreitol  (Roche)


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    Structured Review

    Roche dithiothreitol
    CO oxidation to CO 2 does not drive Rev-Erbβ LBD reduction. GCMS chromatogram of gaseous 13 CO 2 (red) and 12 CO 2 (blue) in the headspace of vials containing a , Labelled CO; b , Buffer containing 10 µM low potential redox mediator dyes; c , Rev-Erbβ (100 μM) and low potential redox mediator dyes (10 μM); d , CODH (4.8 µM), low potential redox mediator dyes (10 µM), <t>dithiothreitol</t> (1 mM).
    Dithiothreitol, supplied by Roche, used in various techniques. Bioz Stars score: 92/100, based on 431 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/dithiothreitol/product/Roche
    Average 92 stars, based on 431 article reviews
    Price from $9.99 to $1999.99
    dithiothreitol - by Bioz Stars, 2020-09
    92/100 stars

    Images

    1) Product Images from "Ferric Heme as a CO/NO Sensor in the Nuclear Receptor Reverbβ by Coupling Gas binding to Electron Transfer"

    Article Title: Ferric Heme as a CO/NO Sensor in the Nuclear Receptor Reverbβ by Coupling Gas binding to Electron Transfer

    Journal: bioRxiv

    doi: 10.1101/2020.06.22.164806

    CO oxidation to CO 2 does not drive Rev-Erbβ LBD reduction. GCMS chromatogram of gaseous 13 CO 2 (red) and 12 CO 2 (blue) in the headspace of vials containing a , Labelled CO; b , Buffer containing 10 µM low potential redox mediator dyes; c , Rev-Erbβ (100 μM) and low potential redox mediator dyes (10 μM); d , CODH (4.8 µM), low potential redox mediator dyes (10 µM), dithiothreitol (1 mM).
    Figure Legend Snippet: CO oxidation to CO 2 does not drive Rev-Erbβ LBD reduction. GCMS chromatogram of gaseous 13 CO 2 (red) and 12 CO 2 (blue) in the headspace of vials containing a , Labelled CO; b , Buffer containing 10 µM low potential redox mediator dyes; c , Rev-Erbβ (100 μM) and low potential redox mediator dyes (10 μM); d , CODH (4.8 µM), low potential redox mediator dyes (10 µM), dithiothreitol (1 mM).

    Techniques Used:

    Related Articles

    Centrifugation:

    Article Title: Ferric Heme as a CO/NO Sensor in the Nuclear Receptor Reverbβ by Coupling Gas binding to Electron Transfer
    Article Snippet: .. The cell pellet obtained by centrifugation was suspended in TNG buffer (50 mM Tris-HCl, pH 8.0, 300 mM NaCl, and 10% glycerol) containing 1 mM dithiothreitol, 6 mM benzamidine, 1 mM EDTA, 0.5 mM phenylmethanesulfonyl fluoride, and 1x protease inhibitor mixture (Roche Applied Science) on ice and lysed by sonication. .. The cell-free extract obtained post centrifugation was applied to an amylose column (New England Biolabs) equilibrated in TNG with 1 mM DTT at 4 °C.

    Article Title: Epidermal Growth Factor-Dependent Phosphorylation and Ubiquitinylation of MAGE-11 Regulates Its Interaction with the Androgen Receptor ▿
    Article Snippet: .. Cells were pelleted by centrifugation and solubilized in 1 ml immunoprecipitation (IP) lysis buffer containing 0.15 M NaCl, 0.5% NP-40, 50 mM NaF, 50 mM Tris-HCl, pH 7.5, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, and protease inhibitor cocktail (Roche). .. Protein extracts were precleared with 100 μl agarose (Sigma), and extracts were incubated with 15 μl anti-Flag M2 affinity gel (Sigma) for 1 to 2 h or overnight at 4°C and washed four times with IP lysis buffer.

    Article Title: Restricting Conformational Flexibility of the Switch II Region Creates a Dominant-Inhibitory Phenotype in Obg GTPase Nog1 ▿
    Article Snippet: .. Bacteria were collected by centrifugation, frozen in pellets, resuspended in 25 mM Tris-HCl (pH 7.4), 100 mM KOAc, 10% glycerol, 1 mM EDTA, 10 mM dithiothreitol, and protease inhibitors (Roche) and lysed by adding Igepal CA-630 to 0.2%. .. After 20 min at 4°C, 10 mM MgOAc2 , 1 mM CaCl2 and DNase I were added to reduce viscosity for 10 min and the lysate was clarified at 10,000 × g for 15 min and filtered through a 0.22-μm filter.

    Protease Inhibitor:

    Article Title: Ferric Heme as a CO/NO Sensor in the Nuclear Receptor Reverbβ by Coupling Gas binding to Electron Transfer
    Article Snippet: .. The cell pellet obtained by centrifugation was suspended in TNG buffer (50 mM Tris-HCl, pH 8.0, 300 mM NaCl, and 10% glycerol) containing 1 mM dithiothreitol, 6 mM benzamidine, 1 mM EDTA, 0.5 mM phenylmethanesulfonyl fluoride, and 1x protease inhibitor mixture (Roche Applied Science) on ice and lysed by sonication. .. The cell-free extract obtained post centrifugation was applied to an amylose column (New England Biolabs) equilibrated in TNG with 1 mM DTT at 4 °C.

    Article Title: Luteinizing hormone signaling phosphorylates and activates the cyclic GMP phosphodiesterase PDE5 in mouse ovarian follicles, contributing an additional component to the hormonally induced decrease in cyclic GMP that reinitiates meiosis
    Article Snippet: .. Equal numbers of follicles (11–17 per experiment) were treated with either LH or control vehicle (PBS) for 30 minutes, and then lysed by probe sonication in 60 μl Laemmli sample buffer with 75 mM dithiothreitol, 10 mM sodium fluoride, 1 mM sodium orthovanadate, 1 mM Pefabloc (Roche Diagnostics, Indianapolis, IN), and 1X Complete Protease Inhibitor, EDTA-free (Roche Diagnostics). .. Lysates were stored at −80°C until the entire 60 μl volume was run on a gel containing 25 μM Phos-tag-acrylamide (Wako Chemicals USA, Richmond, VA) as previously described ( ; ).

    Article Title: Epidermal Growth Factor-Dependent Phosphorylation and Ubiquitinylation of MAGE-11 Regulates Its Interaction with the Androgen Receptor ▿
    Article Snippet: .. Cells were pelleted by centrifugation and solubilized in 1 ml immunoprecipitation (IP) lysis buffer containing 0.15 M NaCl, 0.5% NP-40, 50 mM NaF, 50 mM Tris-HCl, pH 7.5, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, and protease inhibitor cocktail (Roche). .. Protein extracts were precleared with 100 μl agarose (Sigma), and extracts were incubated with 15 μl anti-Flag M2 affinity gel (Sigma) for 1 to 2 h or overnight at 4°C and washed four times with IP lysis buffer.

    Article Title: The Initial Enzyme for Glycosylphosphatidylinositol Biosynthesis Requires PIG-Y, a Seventh Component
    Article Snippet: .. The cell lysate and the protein complex were incubated in 100 μl of a reaction mixture consisting of 2 μCi of UDP-6 [3 H]GlcNAc (American Radiolabeled Chemicals, St. Louis, MO), Bovine PI (100 μM; Sigma; only for protein complexes), 50 mM HEPES-NaOH (pH 7.4), 25 mM KCl, 5 mM MgCl2 , 1 mM ATP, 0.5 mM dithiothreitol, 0.2 μg/ml tunicamycin, and protease inhibitor cocktail, Complete EDTA free (Roche, Indianapolis, IN) for 2 h at 37°C. ..

    Article Title: Protein prenylation and Hsp40 in thermotolerance of Plasmodium falciparum malaria parasites
    Article Snippet: .. Expressed proteins were purified from cells using a sonication lysis buffer containing 1 mg/ml lysozyme, 20mM imidzazole, 1mM dithiothreitol, 1mM MgCl2, 10mM Tris HCl (pH7.5), 30 U benzonase, 1mM phenylmethylsulfonyl fluoride (PMSF), and cOmplete EDTA-free protease inhibitor tablets (Roche, Basel, Switzerland). .. Lysates were clarified using centrifugation and proteins were purified via nickel agarose beads (Gold Biotechnology, Olivette, MO), eluted with 300mM imidazole, 20mM Tris-HCl (pH 7.5) and 150mM NaCl.

    Protein Extraction:

    Article Title: Generation and Characterization of LANP/pp32 Null Mice
    Article Snippet: .. Briefly tissues were homogenized in protein extraction buffer containing 100 mM Tris, pH 6.8, 2% sodium dodecyl sulfate (SDS), 25 mM dithiothreitol, and protease inhibitors (Complete; Roche Molecular Biochemicals). .. Proteins were separated on SDS-10% polyacrylamide gel electrophoresis gels and transferred to nitrocellulose membranes (Schleicher & Schuell).

    Purification:

    Article Title: Protein prenylation and Hsp40 in thermotolerance of Plasmodium falciparum malaria parasites
    Article Snippet: .. Expressed proteins were purified from cells using a sonication lysis buffer containing 1 mg/ml lysozyme, 20mM imidzazole, 1mM dithiothreitol, 1mM MgCl2, 10mM Tris HCl (pH7.5), 30 U benzonase, 1mM phenylmethylsulfonyl fluoride (PMSF), and cOmplete EDTA-free protease inhibitor tablets (Roche, Basel, Switzerland). .. Lysates were clarified using centrifugation and proteins were purified via nickel agarose beads (Gold Biotechnology, Olivette, MO), eluted with 300mM imidazole, 20mM Tris-HCl (pH 7.5) and 150mM NaCl.

    Immunoprecipitation:

    Article Title: Epidermal Growth Factor-Dependent Phosphorylation and Ubiquitinylation of MAGE-11 Regulates Its Interaction with the Androgen Receptor ▿
    Article Snippet: .. Cells were pelleted by centrifugation and solubilized in 1 ml immunoprecipitation (IP) lysis buffer containing 0.15 M NaCl, 0.5% NP-40, 50 mM NaF, 50 mM Tris-HCl, pH 7.5, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, and protease inhibitor cocktail (Roche). .. Protein extracts were precleared with 100 μl agarose (Sigma), and extracts were incubated with 15 μl anti-Flag M2 affinity gel (Sigma) for 1 to 2 h or overnight at 4°C and washed four times with IP lysis buffer.

    Incubation:

    Article Title: The Initial Enzyme for Glycosylphosphatidylinositol Biosynthesis Requires PIG-Y, a Seventh Component
    Article Snippet: .. The cell lysate and the protein complex were incubated in 100 μl of a reaction mixture consisting of 2 μCi of UDP-6 [3 H]GlcNAc (American Radiolabeled Chemicals, St. Louis, MO), Bovine PI (100 μM; Sigma; only for protein complexes), 50 mM HEPES-NaOH (pH 7.4), 25 mM KCl, 5 mM MgCl2 , 1 mM ATP, 0.5 mM dithiothreitol, 0.2 μg/ml tunicamycin, and protease inhibitor cocktail, Complete EDTA free (Roche, Indianapolis, IN) for 2 h at 37°C. ..

    Lysis:

    Article Title: Epidermal Growth Factor-Dependent Phosphorylation and Ubiquitinylation of MAGE-11 Regulates Its Interaction with the Androgen Receptor ▿
    Article Snippet: .. Cells were pelleted by centrifugation and solubilized in 1 ml immunoprecipitation (IP) lysis buffer containing 0.15 M NaCl, 0.5% NP-40, 50 mM NaF, 50 mM Tris-HCl, pH 7.5, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, and protease inhibitor cocktail (Roche). .. Protein extracts were precleared with 100 μl agarose (Sigma), and extracts were incubated with 15 μl anti-Flag M2 affinity gel (Sigma) for 1 to 2 h or overnight at 4°C and washed four times with IP lysis buffer.

    Article Title: Protein prenylation and Hsp40 in thermotolerance of Plasmodium falciparum malaria parasites
    Article Snippet: .. Expressed proteins were purified from cells using a sonication lysis buffer containing 1 mg/ml lysozyme, 20mM imidzazole, 1mM dithiothreitol, 1mM MgCl2, 10mM Tris HCl (pH7.5), 30 U benzonase, 1mM phenylmethylsulfonyl fluoride (PMSF), and cOmplete EDTA-free protease inhibitor tablets (Roche, Basel, Switzerland). .. Lysates were clarified using centrifugation and proteins were purified via nickel agarose beads (Gold Biotechnology, Olivette, MO), eluted with 300mM imidazole, 20mM Tris-HCl (pH 7.5) and 150mM NaCl.

    Sonication:

    Article Title: Ferric Heme as a CO/NO Sensor in the Nuclear Receptor Reverbβ by Coupling Gas binding to Electron Transfer
    Article Snippet: .. The cell pellet obtained by centrifugation was suspended in TNG buffer (50 mM Tris-HCl, pH 8.0, 300 mM NaCl, and 10% glycerol) containing 1 mM dithiothreitol, 6 mM benzamidine, 1 mM EDTA, 0.5 mM phenylmethanesulfonyl fluoride, and 1x protease inhibitor mixture (Roche Applied Science) on ice and lysed by sonication. .. The cell-free extract obtained post centrifugation was applied to an amylose column (New England Biolabs) equilibrated in TNG with 1 mM DTT at 4 °C.

    Article Title: Luteinizing hormone signaling phosphorylates and activates the cyclic GMP phosphodiesterase PDE5 in mouse ovarian follicles, contributing an additional component to the hormonally induced decrease in cyclic GMP that reinitiates meiosis
    Article Snippet: .. Equal numbers of follicles (11–17 per experiment) were treated with either LH or control vehicle (PBS) for 30 minutes, and then lysed by probe sonication in 60 μl Laemmli sample buffer with 75 mM dithiothreitol, 10 mM sodium fluoride, 1 mM sodium orthovanadate, 1 mM Pefabloc (Roche Diagnostics, Indianapolis, IN), and 1X Complete Protease Inhibitor, EDTA-free (Roche Diagnostics). .. Lysates were stored at −80°C until the entire 60 μl volume was run on a gel containing 25 μM Phos-tag-acrylamide (Wako Chemicals USA, Richmond, VA) as previously described ( ; ).

    Article Title: Protein prenylation and Hsp40 in thermotolerance of Plasmodium falciparum malaria parasites
    Article Snippet: .. Expressed proteins were purified from cells using a sonication lysis buffer containing 1 mg/ml lysozyme, 20mM imidzazole, 1mM dithiothreitol, 1mM MgCl2, 10mM Tris HCl (pH7.5), 30 U benzonase, 1mM phenylmethylsulfonyl fluoride (PMSF), and cOmplete EDTA-free protease inhibitor tablets (Roche, Basel, Switzerland). .. Lysates were clarified using centrifugation and proteins were purified via nickel agarose beads (Gold Biotechnology, Olivette, MO), eluted with 300mM imidazole, 20mM Tris-HCl (pH 7.5) and 150mM NaCl.

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  • 94
    Roche dithiothreitol dtt
    Critical concentration measurement of the actin filament. Recombinant actin (0.5–3 µM as final concentrations) was polymerized in F-buffer (2 mM Tris-HCl, 100 mM KCl, 2 mM MgCl 2 , 0.2 mM ATP (adenosine triphosphate), 0.2 mM <t>DTT</t> <t>(dithiothreitol),</t> pH 8.0) by incubating for 60 min at room temperature and separating by ultracentrifugation into the pellet (ppt) and supernatant (sup). An example of an SDS-PAGE gel for wild-type (WT) and the G42A/G46A mutant (AA) is shown. The critical concentration was measured by densitometry of actin in the supernatant. Four independent experiments were performed and the ratio of the critical concentration of the mutant against the WT was 0.82 ± 0.03 (standard error). The left and right lanes are molecular mass markers with molecular weights provided in kilo-Daltons on the left-hand side.
    Dithiothreitol Dtt, supplied by Roche, used in various techniques. Bioz Stars score: 94/100, based on 100 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/dithiothreitol dtt/product/Roche
    Average 94 stars, based on 100 article reviews
    Price from $9.99 to $1999.99
    dithiothreitol dtt - by Bioz Stars, 2020-09
    94/100 stars
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    92
    Roche dithiothreitol
    CO oxidation to CO 2 does not drive Rev-Erbβ LBD reduction. GCMS chromatogram of gaseous 13 CO 2 (red) and 12 CO 2 (blue) in the headspace of vials containing a , Labelled CO; b , Buffer containing 10 µM low potential redox mediator dyes; c , Rev-Erbβ (100 μM) and low potential redox mediator dyes (10 μM); d , CODH (4.8 µM), low potential redox mediator dyes (10 µM), <t>dithiothreitol</t> (1 mM).
    Dithiothreitol, supplied by Roche, used in various techniques. Bioz Stars score: 92/100, based on 431 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/dithiothreitol/product/Roche
    Average 92 stars, based on 431 article reviews
    Price from $9.99 to $1999.99
    dithiothreitol - by Bioz Stars, 2020-09
    92/100 stars
      Buy from Supplier

    85
    Roche m dithiothreitol buffer
    Removal of Zn 2+ causes loss of structure in parkin. CD spectra of full-length parkin (3 μ m ) in 5 m m Tris, 20 m m NaCl, and 1 m m <t>dithiothreitol</t> at pH 7.4. The spectra show full-length parkin in the absence ( thick solid line ), and presence of
    M Dithiothreitol Buffer, supplied by Roche, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/m dithiothreitol buffer/product/Roche
    Average 85 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    m dithiothreitol buffer - by Bioz Stars, 2020-09
    85/100 stars
      Buy from Supplier

    Image Search Results


    Critical concentration measurement of the actin filament. Recombinant actin (0.5–3 µM as final concentrations) was polymerized in F-buffer (2 mM Tris-HCl, 100 mM KCl, 2 mM MgCl 2 , 0.2 mM ATP (adenosine triphosphate), 0.2 mM DTT (dithiothreitol), pH 8.0) by incubating for 60 min at room temperature and separating by ultracentrifugation into the pellet (ppt) and supernatant (sup). An example of an SDS-PAGE gel for wild-type (WT) and the G42A/G46A mutant (AA) is shown. The critical concentration was measured by densitometry of actin in the supernatant. Four independent experiments were performed and the ratio of the critical concentration of the mutant against the WT was 0.82 ± 0.03 (standard error). The left and right lanes are molecular mass markers with molecular weights provided in kilo-Daltons on the left-hand side.

    Journal: Biomolecules

    Article Title: D-Loop Mutation G42A/G46A Decreases Actin Dynamics

    doi: 10.3390/biom10050736

    Figure Lengend Snippet: Critical concentration measurement of the actin filament. Recombinant actin (0.5–3 µM as final concentrations) was polymerized in F-buffer (2 mM Tris-HCl, 100 mM KCl, 2 mM MgCl 2 , 0.2 mM ATP (adenosine triphosphate), 0.2 mM DTT (dithiothreitol), pH 8.0) by incubating for 60 min at room temperature and separating by ultracentrifugation into the pellet (ppt) and supernatant (sup). An example of an SDS-PAGE gel for wild-type (WT) and the G42A/G46A mutant (AA) is shown. The critical concentration was measured by densitometry of actin in the supernatant. Four independent experiments were performed and the ratio of the critical concentration of the mutant against the WT was 0.82 ± 0.03 (standard error). The left and right lanes are molecular mass markers with molecular weights provided in kilo-Daltons on the left-hand side.

    Article Snippet: Purification of Recombinant Actins The infected cells were lysed in extraction buffer (20 mM Tris-HCl, 0.2 mM CaCl2 , 0.2 mM adenosine triphosphate (ATP), 1 mM dithiothreitol (DTT), complete EDTA-free Protease Inhibitor Cocktail (Roche Applied Science, Penzberg, Germany), pH 8.0).

    Techniques: Concentration Assay, Recombinant, SDS Page, Mutagenesis

    CO oxidation to CO 2 does not drive Rev-Erbβ LBD reduction. GCMS chromatogram of gaseous 13 CO 2 (red) and 12 CO 2 (blue) in the headspace of vials containing a , Labelled CO; b , Buffer containing 10 µM low potential redox mediator dyes; c , Rev-Erbβ (100 μM) and low potential redox mediator dyes (10 μM); d , CODH (4.8 µM), low potential redox mediator dyes (10 µM), dithiothreitol (1 mM).

    Journal: bioRxiv

    Article Title: Ferric Heme as a CO/NO Sensor in the Nuclear Receptor Reverbβ by Coupling Gas binding to Electron Transfer

    doi: 10.1101/2020.06.22.164806

    Figure Lengend Snippet: CO oxidation to CO 2 does not drive Rev-Erbβ LBD reduction. GCMS chromatogram of gaseous 13 CO 2 (red) and 12 CO 2 (blue) in the headspace of vials containing a , Labelled CO; b , Buffer containing 10 µM low potential redox mediator dyes; c , Rev-Erbβ (100 μM) and low potential redox mediator dyes (10 μM); d , CODH (4.8 µM), low potential redox mediator dyes (10 µM), dithiothreitol (1 mM).

    Article Snippet: The cell pellet obtained by centrifugation was suspended in TNG buffer (50 mM Tris-HCl, pH 8.0, 300 mM NaCl, and 10% glycerol) containing 1 mM dithiothreitol, 6 mM benzamidine, 1 mM EDTA, 0.5 mM phenylmethanesulfonyl fluoride, and 1x protease inhibitor mixture (Roche Applied Science) on ice and lysed by sonication.

    Techniques:

    Concentration-dependent autoactivation αPAK. (A) Flag-αPAK at 0.5 or 2.5 μM as indicated was immobilized on 20 μl of anti-Flag M2 beads (corresponding to 10 or 50 μl of COS7 total cell lysates from two different experiments as indicated). The beads were incubated with 200 μM ATP in a solution containing 50 mM HEPES (pH 7.3), 10 mM MgCl 2 , 1 mM dithiothreitol, and 0.05% Triton X-100 for 15 min at 37°C, and the reaction was quenched by adding SDS-PAGE sample buffer. Samples were diluted to the same final concentration of αPAK (40 or 200 μl, respectively) for Western blot analysis with anti-phospho-S198/203 or anti-αPAK as shown. The concentration of αPAK was determined by Coomassie blue staining relative to bovine serum albumin standard. The increased immunoreactivity at higher protein concentration was quantified (as indicated) by densitometry of the bands: anti-phospho S198/203 signal at 0.5 μM αPAK is defined as 1. (B) PAK autophosphorylation does not require intact CRIB sequence. Different concentrations of Flag-αPAK or Flag-αPAK(S76P) were immobilized and allowed to undergo autophosphorylation under conditions described for A. To assess Cdc42-mediated activation, recombinant GST-Cdc42V12 (1 μM) was added with the same ATP-containing buffer and incubated as described for panel A (15 min at 37°C). Reactions were quenched by adding SDS-PAGE sample buffer. Note the appearance of lower-mobility species in parallel with anti-phosphoS198/203 signal.

    Journal: Molecular and Cellular Biology

    Article Title: GIT1 Activates p21-Activated Kinase through a Mechanism Independent of p21 Binding

    doi: 10.1128/MCB.24.9.3849-3859.2004

    Figure Lengend Snippet: Concentration-dependent autoactivation αPAK. (A) Flag-αPAK at 0.5 or 2.5 μM as indicated was immobilized on 20 μl of anti-Flag M2 beads (corresponding to 10 or 50 μl of COS7 total cell lysates from two different experiments as indicated). The beads were incubated with 200 μM ATP in a solution containing 50 mM HEPES (pH 7.3), 10 mM MgCl 2 , 1 mM dithiothreitol, and 0.05% Triton X-100 for 15 min at 37°C, and the reaction was quenched by adding SDS-PAGE sample buffer. Samples were diluted to the same final concentration of αPAK (40 or 200 μl, respectively) for Western blot analysis with anti-phospho-S198/203 or anti-αPAK as shown. The concentration of αPAK was determined by Coomassie blue staining relative to bovine serum albumin standard. The increased immunoreactivity at higher protein concentration was quantified (as indicated) by densitometry of the bands: anti-phospho S198/203 signal at 0.5 μM αPAK is defined as 1. (B) PAK autophosphorylation does not require intact CRIB sequence. Different concentrations of Flag-αPAK or Flag-αPAK(S76P) were immobilized and allowed to undergo autophosphorylation under conditions described for A. To assess Cdc42-mediated activation, recombinant GST-Cdc42V12 (1 μM) was added with the same ATP-containing buffer and incubated as described for panel A (15 min at 37°C). Reactions were quenched by adding SDS-PAGE sample buffer. Note the appearance of lower-mobility species in parallel with anti-phosphoS198/203 signal.

    Article Snippet: Transfected cells were harvested by scraping in ice cold cell lysis buffer (500 μl of 50 mM HEPES [pH 7.3], 150 mM NaCl, 1.5 mM MgCl2 , 1 mM EDTA, 20 mM β-glycerophosphate, 5% glycerol, 1% Triton X-100, 1 mM dithiothreitol, and a protease inhibitor cocktail [Roche]).

    Techniques: Concentration Assay, Incubation, SDS Page, Western Blot, Staining, Protein Concentration, Sequencing, Activation Assay, Recombinant

    Removal of Zn 2+ causes loss of structure in parkin. CD spectra of full-length parkin (3 μ m ) in 5 m m Tris, 20 m m NaCl, and 1 m m dithiothreitol at pH 7.4. The spectra show full-length parkin in the absence ( thick solid line ), and presence of

    Journal: The Journal of Biological Chemistry

    Article Title: Identification of a Novel Zn2+-binding Domain in the Autosomal Recessive Juvenile Parkinson-related E3 Ligase Parkin *-binding Domain in the Autosomal Recessive Juvenile Parkinson-related E3 Ligase Parkin * S⃞

    doi: 10.1074/jbc.M808700200

    Figure Lengend Snippet: Removal of Zn 2+ causes loss of structure in parkin. CD spectra of full-length parkin (3 μ m ) in 5 m m Tris, 20 m m NaCl, and 1 m m dithiothreitol at pH 7.4. The spectra show full-length parkin in the absence ( thick solid line ), and presence of

    Article Snippet: The E. coli cells were harvested and lysed by sonication on ice in 20 m m Tris-HCl (pH 7.4), 120 m m NaCl, 1 m m dithiothreitol buffer supplemented with EDTA-free protease inhibitor mixture (Roche Applied Science).

    Techniques: