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Bio-Rad phenylmethylsulfonyl fluoride
Effects of thiol reagents on the ACCase activity. ( A ) ACCase activity was measured in the indicated concentration of a thiol reagent as described in Materials and Methods . Plastidic ACCase from pea, continuous lines; cytosolic ACCase from pea, dashed line. β-SH, 2-mercaptoethanol; GSH, reduced form of glutathione. Lipoic acid was dissolved into 50 mM Tricine⋅KOH (pH 8) containing 1 mM <t>phenylmethylsulfonyl</t> fluoride, 1 mM benzamide, and 5 mM ɛ-amino-n-caproic acid at 20 mM, and 0–7 μl of 20 mM solution was added to 13-μl reaction mixture. The activity without any thiol reagent was 11 nmol/20 min per mg of protein for pea plastidic ACCase and 210 nmol/20 min per mg of protein for pea cytosolic ACCase. The activity is relative to that without a thiol reagent, taken to be 1. Results are the means of duplicate determinations that differed less than 10%. ( B ) The effects of preincubation of plastidic ACCase with DTT on activity were examined. Changes with time are shown. After the enzyme was incubated at 30°C for 10 min with 2 mM DTT, the reaction was started by the addition of buffer, ATP, MgCl 2 , KCl, acetyl-CoA, and NaH 14 CO 3 . The final concentrations of these reagents were the same as above. The final concentration of DTT was 1 mM.
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1) Product Images from "Link between light and fatty acid synthesis: Thioredoxin-linked reductive activation of plastidic acetyl-CoA carboxylase"

Article Title: Link between light and fatty acid synthesis: Thioredoxin-linked reductive activation of plastidic acetyl-CoA carboxylase

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi:

Effects of thiol reagents on the ACCase activity. ( A ) ACCase activity was measured in the indicated concentration of a thiol reagent as described in Materials and Methods . Plastidic ACCase from pea, continuous lines; cytosolic ACCase from pea, dashed line. β-SH, 2-mercaptoethanol; GSH, reduced form of glutathione. Lipoic acid was dissolved into 50 mM Tricine⋅KOH (pH 8) containing 1 mM phenylmethylsulfonyl fluoride, 1 mM benzamide, and 5 mM ɛ-amino-n-caproic acid at 20 mM, and 0–7 μl of 20 mM solution was added to 13-μl reaction mixture. The activity without any thiol reagent was 11 nmol/20 min per mg of protein for pea plastidic ACCase and 210 nmol/20 min per mg of protein for pea cytosolic ACCase. The activity is relative to that without a thiol reagent, taken to be 1. Results are the means of duplicate determinations that differed less than 10%. ( B ) The effects of preincubation of plastidic ACCase with DTT on activity were examined. Changes with time are shown. After the enzyme was incubated at 30°C for 10 min with 2 mM DTT, the reaction was started by the addition of buffer, ATP, MgCl 2 , KCl, acetyl-CoA, and NaH 14 CO 3 . The final concentrations of these reagents were the same as above. The final concentration of DTT was 1 mM.
Figure Legend Snippet: Effects of thiol reagents on the ACCase activity. ( A ) ACCase activity was measured in the indicated concentration of a thiol reagent as described in Materials and Methods . Plastidic ACCase from pea, continuous lines; cytosolic ACCase from pea, dashed line. β-SH, 2-mercaptoethanol; GSH, reduced form of glutathione. Lipoic acid was dissolved into 50 mM Tricine⋅KOH (pH 8) containing 1 mM phenylmethylsulfonyl fluoride, 1 mM benzamide, and 5 mM ɛ-amino-n-caproic acid at 20 mM, and 0–7 μl of 20 mM solution was added to 13-μl reaction mixture. The activity without any thiol reagent was 11 nmol/20 min per mg of protein for pea plastidic ACCase and 210 nmol/20 min per mg of protein for pea cytosolic ACCase. The activity is relative to that without a thiol reagent, taken to be 1. Results are the means of duplicate determinations that differed less than 10%. ( B ) The effects of preincubation of plastidic ACCase with DTT on activity were examined. Changes with time are shown. After the enzyme was incubated at 30°C for 10 min with 2 mM DTT, the reaction was started by the addition of buffer, ATP, MgCl 2 , KCl, acetyl-CoA, and NaH 14 CO 3 . The final concentrations of these reagents were the same as above. The final concentration of DTT was 1 mM.

Techniques Used: Activity Assay, Concentration Assay, Incubation

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Radio Immunoprecipitation:

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Article Snippet: .. Whole cells were lysed in radioimmunoprecipitation assay (RIPA) buffer (10 mM Tris-HCl [pH 7.4], 0.15 M NaCl, 0.5% SDS, 1% NP-40, 1% Na-deoxycholate, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride [PMSF], 1 μg/ml pepstatin, and 1 μg/ml leupeptin), and the protein concentration was determined by using the Bradford protein assay kit (Bio-Rad, CA). ..

Protein Concentration:

Article Title: The Gene for Aromatase, a Rate-Limiting Enzyme for Local Estrogen Biosynthesis, Is a Downstream Target Gene of Runx2 in Skeletal Tissues ▿
Article Snippet: .. Whole cells were lysed in radioimmunoprecipitation assay (RIPA) buffer (10 mM Tris-HCl [pH 7.4], 0.15 M NaCl, 0.5% SDS, 1% NP-40, 1% Na-deoxycholate, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride [PMSF], 1 μg/ml pepstatin, and 1 μg/ml leupeptin), and the protein concentration was determined by using the Bradford protein assay kit (Bio-Rad, CA). ..

Bradford Protein Assay:

Article Title: The Gene for Aromatase, a Rate-Limiting Enzyme for Local Estrogen Biosynthesis, Is a Downstream Target Gene of Runx2 in Skeletal Tissues ▿
Article Snippet: .. Whole cells were lysed in radioimmunoprecipitation assay (RIPA) buffer (10 mM Tris-HCl [pH 7.4], 0.15 M NaCl, 0.5% SDS, 1% NP-40, 1% Na-deoxycholate, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride [PMSF], 1 μg/ml pepstatin, and 1 μg/ml leupeptin), and the protein concentration was determined by using the Bradford protein assay kit (Bio-Rad, CA). ..

Protease Inhibitor:

Article Title: The prrAB Two-Component System Is Essential for Mycobacterium tuberculosis Viability and Is Induced under Nitrogen-Limiting Conditions
Article Snippet: To ensure complete stripping of the DIG-labeled probe before subsequent hybridizations, membranes were incubated twice in a 50% formamide solution containing 50 mM Tris-HCl, pH 7.5, and 5% SDS at 80°C for 60 min and then subjected to autoradiography. .. M. tuberculosis cells were washed twice in wash buffer (phosphate-buffered saline [PBS] buffer containing protease inhibitor cocktail [Roche], 1 mM dithiothreitol [DTT], 1 mM EDTA, and 1 mM phenylmethylsulfonyl fluoride [PMSF]) and lysed in wash buffer or two-dimensional (2D)-rehydration buffer (Bio-Rad Laboratories) with 0.1-mm-diameter zirconia/silica beads (Biospec Products) in a Fast Prep homogenizer. ..

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Article Title: Low Density Lipoprotein Binds to Proprotein Convertase Subtilisin/Kexin Type-9 (PCSK9) in Human Plasma and Inhibits PCSK9-mediated Low Density Lipoprotein Receptor Degradation *
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SDS Page:

Article Title: Inverse regulation of Vibrio cholerae biofilm dispersal by polyamine signals
Article Snippet: Western blotting Cultures of strains carrying MbaA-3xFLAG (and relevant catalytic site variants) were collected at OD600 = 1.0 and subjected to centrifugation for 1 min at 13,000 rpm. .. The pellets were flash frozen, thawed for 5 min at 25° C, and subsequently chemically lysed by resuspending to OD600 = 1.0 in 75 μL Bug Buster (Novagen, #70584–4) supplemented with 0.5% Triton-X, 50 μg/mL lysozyme, 25 U/mL benzonase nuclease, and 1 mM phenylmethylsulfonyl fluoride (PMSF) for 10 min at 25° C. Lysates were solubilized in 1X SDS-PAGE buffer for 1 h at 37° C. Samples were loaded into 4–20% Mini-Protein TGX gels (Bio-Rad). .. Electrophoresis was carried out at 200 V. Proteins were transferred from the gels to PVDF membranes (Bio-Rad) for 50 min at 4° C at 100 V in 25 mM Tris base, 190 mM glycine, 20% methanol.

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Article Title: Low Density Lipoprotein Binds to Proprotein Convertase Subtilisin/Kexin Type-9 (PCSK9) in Human Plasma and Inhibits PCSK9-mediated Low Density Lipoprotein Receptor Degradation *
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Lysis:

Article Title: Low Density Lipoprotein Binds to Proprotein Convertase Subtilisin/Kexin Type-9 (PCSK9) in Human Plasma and Inhibits PCSK9-mediated Low Density Lipoprotein Receptor Degradation *
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    Bio-Rad pmsf
    Detection of CheA1 in different subcellular fractions of A. brasilense wild-type strain Sp7 and its Δ cheA1 mutant derivative by Western blotting. (A) Whole-cell lysates of the wild type and the Δ cheA1 mutant were probed with affinity-purified polyclonal antibodies raised against a recombinantly produced soluble CheA1ΔTMX from A. brasilense Sp7, as described in Materials and Methods. (B) Different subcellular fractions collected from the wild-type strain Sp7 were probed with affinity-purified polyclonal antibodies raised against a recombinantly produced soluble CheA1ΔTMX from A. brasilense Sp7. Note that different protocols were used for sample preparation and SDS-PAGE for the Western blots shown in panels A and B (see Materials and Methods). In panel A, an anti-CheAΔTMX antibody, which was further purified against whole proteins extracted from the Δ che1 mutant, was used. These samples were run on a 7.5% gel and transferred to a 0.45-μm hydrophobic PVDF transfer membrane. In panel B, an anti-CheAΔTMX antibody which had not been further purified against whole proteins extracted from the Δ che1 mutant was used. The membrane fraction was <t>resuspended</t> in a solubilizing buffer (1× PBS with 2% Triton X-100 and 1 mM <t>PMSF)</t> and run on an 8 to 16% gradient gel before being transferred to a nitrocellulose membrane. For the cytosolic fraction, these samples were concentrated via acetone precipitation before being loaded in a 10% gel and transferred to a nitrocellulose membrane. The numbers on the left indicate the positions of the molecular mass markers run on the same gels. The arrows point to the soluble, faint band corresponding to CheA1ΔTMX.
    Pmsf, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bio-Rad phenylmethylsulfonyl fluoride
    Effects of thiol reagents on the ACCase activity. ( A ) ACCase activity was measured in the indicated concentration of a thiol reagent as described in Materials and Methods . Plastidic ACCase from pea, continuous lines; cytosolic ACCase from pea, dashed line. β-SH, 2-mercaptoethanol; GSH, reduced form of glutathione. Lipoic acid was dissolved into 50 mM Tricine⋅KOH (pH 8) containing 1 mM <t>phenylmethylsulfonyl</t> fluoride, 1 mM benzamide, and 5 mM ɛ-amino-n-caproic acid at 20 mM, and 0–7 μl of 20 mM solution was added to 13-μl reaction mixture. The activity without any thiol reagent was 11 nmol/20 min per mg of protein for pea plastidic ACCase and 210 nmol/20 min per mg of protein for pea cytosolic ACCase. The activity is relative to that without a thiol reagent, taken to be 1. Results are the means of duplicate determinations that differed less than 10%. ( B ) The effects of preincubation of plastidic ACCase with DTT on activity were examined. Changes with time are shown. After the enzyme was incubated at 30°C for 10 min with 2 mM DTT, the reaction was started by the addition of buffer, ATP, MgCl 2 , KCl, acetyl-CoA, and NaH 14 CO 3 . The final concentrations of these reagents were the same as above. The final concentration of DTT was 1 mM.
    Phenylmethylsulfonyl Fluoride, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/phenylmethylsulfonyl fluoride/product/Bio-Rad
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    phenylmethylsulfonyl fluoride - by Bioz Stars, 2021-03
    86/100 stars
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    Detection of CheA1 in different subcellular fractions of A. brasilense wild-type strain Sp7 and its Δ cheA1 mutant derivative by Western blotting. (A) Whole-cell lysates of the wild type and the Δ cheA1 mutant were probed with affinity-purified polyclonal antibodies raised against a recombinantly produced soluble CheA1ΔTMX from A. brasilense Sp7, as described in Materials and Methods. (B) Different subcellular fractions collected from the wild-type strain Sp7 were probed with affinity-purified polyclonal antibodies raised against a recombinantly produced soluble CheA1ΔTMX from A. brasilense Sp7. Note that different protocols were used for sample preparation and SDS-PAGE for the Western blots shown in panels A and B (see Materials and Methods). In panel A, an anti-CheAΔTMX antibody, which was further purified against whole proteins extracted from the Δ che1 mutant, was used. These samples were run on a 7.5% gel and transferred to a 0.45-μm hydrophobic PVDF transfer membrane. In panel B, an anti-CheAΔTMX antibody which had not been further purified against whole proteins extracted from the Δ che1 mutant was used. The membrane fraction was resuspended in a solubilizing buffer (1× PBS with 2% Triton X-100 and 1 mM PMSF) and run on an 8 to 16% gradient gel before being transferred to a nitrocellulose membrane. For the cytosolic fraction, these samples were concentrated via acetone precipitation before being loaded in a 10% gel and transferred to a nitrocellulose membrane. The numbers on the left indicate the positions of the molecular mass markers run on the same gels. The arrows point to the soluble, faint band corresponding to CheA1ΔTMX.

    Journal: Journal of Bacteriology

    Article Title: Distinct Domains of CheA Confer Unique Functions in Chemotaxis and Cell Length in Azospirillum brasilense Sp7

    doi: 10.1128/JB.00189-17

    Figure Lengend Snippet: Detection of CheA1 in different subcellular fractions of A. brasilense wild-type strain Sp7 and its Δ cheA1 mutant derivative by Western blotting. (A) Whole-cell lysates of the wild type and the Δ cheA1 mutant were probed with affinity-purified polyclonal antibodies raised against a recombinantly produced soluble CheA1ΔTMX from A. brasilense Sp7, as described in Materials and Methods. (B) Different subcellular fractions collected from the wild-type strain Sp7 were probed with affinity-purified polyclonal antibodies raised against a recombinantly produced soluble CheA1ΔTMX from A. brasilense Sp7. Note that different protocols were used for sample preparation and SDS-PAGE for the Western blots shown in panels A and B (see Materials and Methods). In panel A, an anti-CheAΔTMX antibody, which was further purified against whole proteins extracted from the Δ che1 mutant, was used. These samples were run on a 7.5% gel and transferred to a 0.45-μm hydrophobic PVDF transfer membrane. In panel B, an anti-CheAΔTMX antibody which had not been further purified against whole proteins extracted from the Δ che1 mutant was used. The membrane fraction was resuspended in a solubilizing buffer (1× PBS with 2% Triton X-100 and 1 mM PMSF) and run on an 8 to 16% gradient gel before being transferred to a nitrocellulose membrane. For the cytosolic fraction, these samples were concentrated via acetone precipitation before being loaded in a 10% gel and transferred to a nitrocellulose membrane. The numbers on the left indicate the positions of the molecular mass markers run on the same gels. The arrows point to the soluble, faint band corresponding to CheA1ΔTMX.

    Article Snippet: Equal volumes of the membrane pellet, which was resuspended in 1× PBS with 2% Triton X-100, 1 mM PMSF, and 2× Laemmli buffer with reducing agent, were boiled for 5 min prior to being loaded, at an amount of 31 μg, into an 8 to 16% Mini-Protean precast gradient gel (Bio-Rad).

    Techniques: Mutagenesis, Western Blot, Affinity Purification, Produced, Sample Prep, SDS Page, Purification

    Effects of thiol reagents on the ACCase activity. ( A ) ACCase activity was measured in the indicated concentration of a thiol reagent as described in Materials and Methods . Plastidic ACCase from pea, continuous lines; cytosolic ACCase from pea, dashed line. β-SH, 2-mercaptoethanol; GSH, reduced form of glutathione. Lipoic acid was dissolved into 50 mM Tricine⋅KOH (pH 8) containing 1 mM phenylmethylsulfonyl fluoride, 1 mM benzamide, and 5 mM ɛ-amino-n-caproic acid at 20 mM, and 0–7 μl of 20 mM solution was added to 13-μl reaction mixture. The activity without any thiol reagent was 11 nmol/20 min per mg of protein for pea plastidic ACCase and 210 nmol/20 min per mg of protein for pea cytosolic ACCase. The activity is relative to that without a thiol reagent, taken to be 1. Results are the means of duplicate determinations that differed less than 10%. ( B ) The effects of preincubation of plastidic ACCase with DTT on activity were examined. Changes with time are shown. After the enzyme was incubated at 30°C for 10 min with 2 mM DTT, the reaction was started by the addition of buffer, ATP, MgCl 2 , KCl, acetyl-CoA, and NaH 14 CO 3 . The final concentrations of these reagents were the same as above. The final concentration of DTT was 1 mM.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Link between light and fatty acid synthesis: Thioredoxin-linked reductive activation of plastidic acetyl-CoA carboxylase

    doi:

    Figure Lengend Snippet: Effects of thiol reagents on the ACCase activity. ( A ) ACCase activity was measured in the indicated concentration of a thiol reagent as described in Materials and Methods . Plastidic ACCase from pea, continuous lines; cytosolic ACCase from pea, dashed line. β-SH, 2-mercaptoethanol; GSH, reduced form of glutathione. Lipoic acid was dissolved into 50 mM Tricine⋅KOH (pH 8) containing 1 mM phenylmethylsulfonyl fluoride, 1 mM benzamide, and 5 mM ɛ-amino-n-caproic acid at 20 mM, and 0–7 μl of 20 mM solution was added to 13-μl reaction mixture. The activity without any thiol reagent was 11 nmol/20 min per mg of protein for pea plastidic ACCase and 210 nmol/20 min per mg of protein for pea cytosolic ACCase. The activity is relative to that without a thiol reagent, taken to be 1. Results are the means of duplicate determinations that differed less than 10%. ( B ) The effects of preincubation of plastidic ACCase with DTT on activity were examined. Changes with time are shown. After the enzyme was incubated at 30°C for 10 min with 2 mM DTT, the reaction was started by the addition of buffer, ATP, MgCl 2 , KCl, acetyl-CoA, and NaH 14 CO 3 . The final concentrations of these reagents were the same as above. The final concentration of DTT was 1 mM.

    Article Snippet: The ammonium sulfate precipitates of the plastidic or the cytosolic ACCase fraction were dissolved in 50–100 μl of a 50 mM Tricine⋅KOH (pH 8) buffer containing 1 mM phenylmethylsulfonyl fluoride, 1 mM benzamide, and 5 mM ɛ-amino-n-caproic acid, and desalted by centrifugation in a gel filtration column (Biospin 6, or Biospin 30, Bio-Rad).

    Techniques: Activity Assay, Concentration Assay, Incubation