phenylmethylsulfonyl fluoride  (Millipore)

 
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    Name:
    PMSF
    Description:

    Catalog Number:
    pmsf-ro
    Price:
    None
    Applications:
    PMSF (C7H7O2SF) inhibits serine proteases such as chymotrypsin, trypsin, thrombin, and the cysteine protease papain (reversible by DTT treatment). It does not inhibit metalloproteases, most cysteine proteases, or aspartic proteases.PMSF (phenylmethylsulfonyl fluoride) has been used for the preparation of protein extracts from tissues and cells prior to Western blotting.
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    Structured Review

    Millipore phenylmethylsulfonyl fluoride
    PMSF

    https://www.bioz.com/result/phenylmethylsulfonyl fluoride/product/Millipore
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    phenylmethylsulfonyl fluoride - by Bioz Stars, 2021-03
    99/100 stars

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    Incubation:

    Article Title: Bacillus sp. JR3 esterase LipJ: A new mesophilic enzyme showing traces of a thermophilic past
    Article Snippet: Residual activity was measured at 30°C and pH 7 using a conventional assay on p NP-butyrate, and expressed as a percentage of activity without ions or inhibitors, respectively. .. Supernatant samples of Bacillus sp. JR3 were also incubated in the presence of 1 mM PMSF (phenylmethylsulfonyl fluoride, Sigma) to measure the effect of this serine inhibitor on enzyme activity. .. Kinetic parameters (Vmax and Km) were determined under optimal assay conditions by fitting hyperbolic Michaelis-Menten curves with GraphPad Prism ® software version 6.

    Article Title: A Novel Pathway for Mammary Epithelial Cell Invasion Induced by the Helix-Loop-Helix Protein Id-1
    Article Snippet: Briefly, gels consisted of 8 to 10% polyacrylamide and 3 mg of α-casein or gelatin (Sigma)/ml. .. Concentrated conditioned medium was mixed with nonreducing Laemmli sample buffer and incubated at 37°C for 15 min. After electrophoresis, the gels were incubated for 1 h in 2.5% Triton X-100 at room temperature, followed by 24 to 48 h in substrate buffer (100 mM Tris-HCl [pH 7.4]–15 mM CaCl2 ) in the absence or presence of GM6001 (0.2 mM in DMSO; supplied by Glycomed Corporation and obtained from Z. Werb [ ]), EDTA (10 mM), ortho -phenanthroline (1 mM in DMSO; Sigma), PMSF (5 mM), or AEBSF (0.5 mM; Calbiochem). .. The gels were stained with Coomassie blue for 30 min and were destained with 30% methanol–10% acetic acid.

    Activity Assay:

    Article Title: Bacillus sp. JR3 esterase LipJ: A new mesophilic enzyme showing traces of a thermophilic past
    Article Snippet: Residual activity was measured at 30°C and pH 7 using a conventional assay on p NP-butyrate, and expressed as a percentage of activity without ions or inhibitors, respectively. .. Supernatant samples of Bacillus sp. JR3 were also incubated in the presence of 1 mM PMSF (phenylmethylsulfonyl fluoride, Sigma) to measure the effect of this serine inhibitor on enzyme activity. .. Kinetic parameters (Vmax and Km) were determined under optimal assay conditions by fitting hyperbolic Michaelis-Menten curves with GraphPad Prism ® software version 6.

    Article Title: Ku86 exists as both a full-length and a protease-sensitive natural variant in multiple myeloma cells
    Article Snippet: The reaction mix was incubated at 37°C; and at various time points, aliquots were removed and resolved in a 12.5% SDS-PAGE gel for carbohydrate (CHO) release. .. Intracellular inhibition of protease digestion In order to inhibit intracellular protease activity, 5 × 106 cells/sample were treated with: Complete™ protease inhibitor tablets; either 1× (1 tablet for every 50 mL of media) or 2× (2 tablets for every 50 mL of media); or treated with antipain (2.0 μg/mL) plus leupeptin (2.0 μg/mL) (both from Sigma-Aldrich, St Louis, MO, USA) for cysteine protease inhibition; or aprotinin (2.0 μg/mL) plus PMSF (100 μg/mL) (both from Sigma-Aldrich) for serine protease inhibition; for to 24 hrs. ..

    Lysis:

    Article Title: Main constraints for RNAi induced by expressed long dsRNA in mouse cells
    Article Snippet: Western blotting 3T3 cells were grown in six-well plates. .. Before collection, the cells were washed with PBS and lysed in lysis buffer (20 mM Hepes [pH 7.8], 100 mM NaCl, 1 mM EDTA [pH 8.0], 0.5% IGEPAL-25%, 1 mM fresh DTT, 0.5 mM PMSF, 1 mM NaF, 0.2 mM Na3 VO4 , supplemented with 2× protease inhibitor cocktail set [Millipore], 2× phosphatase inhibitor cocktail set [Millipore], and RiboLock RNase inhibitor [Thermo Fisher Scientific]). .. Proteins were separated on 10% polyacrylamide gel and transferred to a PVFD membrane (Millipore).

    Protease Inhibitor:

    Article Title: Main constraints for RNAi induced by expressed long dsRNA in mouse cells
    Article Snippet: Western blotting 3T3 cells were grown in six-well plates. .. Before collection, the cells were washed with PBS and lysed in lysis buffer (20 mM Hepes [pH 7.8], 100 mM NaCl, 1 mM EDTA [pH 8.0], 0.5% IGEPAL-25%, 1 mM fresh DTT, 0.5 mM PMSF, 1 mM NaF, 0.2 mM Na3 VO4 , supplemented with 2× protease inhibitor cocktail set [Millipore], 2× phosphatase inhibitor cocktail set [Millipore], and RiboLock RNase inhibitor [Thermo Fisher Scientific]). .. Proteins were separated on 10% polyacrylamide gel and transferred to a PVFD membrane (Millipore).

    Article Title: Ku86 exists as both a full-length and a protease-sensitive natural variant in multiple myeloma cells
    Article Snippet: The reaction mix was incubated at 37°C; and at various time points, aliquots were removed and resolved in a 12.5% SDS-PAGE gel for carbohydrate (CHO) release. .. Intracellular inhibition of protease digestion In order to inhibit intracellular protease activity, 5 × 106 cells/sample were treated with: Complete™ protease inhibitor tablets; either 1× (1 tablet for every 50 mL of media) or 2× (2 tablets for every 50 mL of media); or treated with antipain (2.0 μg/mL) plus leupeptin (2.0 μg/mL) (both from Sigma-Aldrich, St Louis, MO, USA) for cysteine protease inhibition; or aprotinin (2.0 μg/mL) plus PMSF (100 μg/mL) (both from Sigma-Aldrich) for serine protease inhibition; for to 24 hrs. ..

    Electrophoresis:

    Article Title: A Novel Pathway for Mammary Epithelial Cell Invasion Induced by the Helix-Loop-Helix Protein Id-1
    Article Snippet: Briefly, gels consisted of 8 to 10% polyacrylamide and 3 mg of α-casein or gelatin (Sigma)/ml. .. Concentrated conditioned medium was mixed with nonreducing Laemmli sample buffer and incubated at 37°C for 15 min. After electrophoresis, the gels were incubated for 1 h in 2.5% Triton X-100 at room temperature, followed by 24 to 48 h in substrate buffer (100 mM Tris-HCl [pH 7.4]–15 mM CaCl2 ) in the absence or presence of GM6001 (0.2 mM in DMSO; supplied by Glycomed Corporation and obtained from Z. Werb [ ]), EDTA (10 mM), ortho -phenanthroline (1 mM in DMSO; Sigma), PMSF (5 mM), or AEBSF (0.5 mM; Calbiochem). .. The gels were stained with Coomassie blue for 30 min and were destained with 30% methanol–10% acetic acid.

    Inhibition:

    Article Title: Ku86 exists as both a full-length and a protease-sensitive natural variant in multiple myeloma cells
    Article Snippet: The reaction mix was incubated at 37°C; and at various time points, aliquots were removed and resolved in a 12.5% SDS-PAGE gel for carbohydrate (CHO) release. .. Intracellular inhibition of protease digestion In order to inhibit intracellular protease activity, 5 × 106 cells/sample were treated with: Complete™ protease inhibitor tablets; either 1× (1 tablet for every 50 mL of media) or 2× (2 tablets for every 50 mL of media); or treated with antipain (2.0 μg/mL) plus leupeptin (2.0 μg/mL) (both from Sigma-Aldrich, St Louis, MO, USA) for cysteine protease inhibition; or aprotinin (2.0 μg/mL) plus PMSF (100 μg/mL) (both from Sigma-Aldrich) for serine protease inhibition; for to 24 hrs. ..

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    Millipore hat assay buffer
    Inhibitory effects of TA on <t>HAT</t> activity. (A) A colorimetric assay was performed to measure HAT activity in the presence of TA at the indicated concentrations. The results are presented as percentages relative to the control sample, which was incubated without TA (left panel). The IC 50 value of TA inhibition of HAT activity was calculated using Prism software (right panel). (B) An in vitro HAT assay using autoradiography was performed to evaluate the effect of the anti-HAT activity of TA on the acetylation of a synthetic H4 peptide by a <t>HeLa</t> NE. The arrow indicates histone H4 (left panel). The autoradiography was quantified using Image J software (right panel). The values presented are the means ± SD of two independent experiments. Means with different superscript letters are significantly different, p
    Hat Assay Buffer, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    97
    Millipore phenylmethylsulfonyl fluoride
    MMP inhibitors prevent the increases in vascular gelatinolytic activity, in ROS concentrations, and in lucigenin chemiluminescence after intraluminal incubation of rabbit aorta with MMP-2. (A) Representative photomicrographs of aortic in situ zymography experiments showing that MMP-2 gelatinolytic activity was totally inhibited by phenanthroline (Phe, 100 µM) or doxycycline (Doxy, 100 µM), but not by <t>phenylmethylsulfonyl</t> fluoride (PMSF, 100 µM). The arteries were incubated with the inhibitors together with MMP-2 (16 nM) intraluminally for 30 min. (B) Quantification of aortic gelatinolytic activity measured in experiments described in A. (C) Representative photomicrographs of aortic sections incubated with DHE. Red fluorescence reflects ROS production and shows that incubation with MMP-2 (16 nM) intraluminally for 30 min increased ROS production, and this effect was totally inhibited by phenanthroline (Phe, 100 µM) or doxycycline (Doxy, 100 µM), but not by phenylmethylsulfonyl fluoride (PMSF, 100 µM). The experiments were carried out as described in A. (D), Quantification of aortic fluorescence of DHE measured in experiments described in C. (E), Quantification of lucigenin chemiluminescence measured in fresh aortas after intraluminal incubation with MMP-2 (16 nM) or vehicle for 30 min in absence or presence of MMP inhibitors, doxycycline (Doxy 100 µM) or GM6001 (10 µM). Data are shown as the mean ± SEM (n = 4–5/group). * P
    Phenylmethylsulfonyl Fluoride, 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/phenylmethylsulfonyl fluoride/product/Millipore
    Average 97 stars, based on 1 article reviews
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    97
    Millipore pmsf
    Expression of a 120-kDa gelatinase by Id-1-expressing mammary epithelial cells. (A) Gelatin zymogram of conditioned media from SCp2–Id-1A (lane 1) and SCp2–Id-1E (lane 2) cells. Cells were cultured in serum-free medium, and conditioned media were harvested and analyzed on a gelatin substrate gel, as described in Materials and Methods. (B) Gelatin zymogram of conditioned media from SCp2–Id-1A cells either growth arrested by serum deprivation (lane 1) or growing in 5% serum (lane 2). (C) Gelatin zymogram of conditioned media from control SCp2 cells (lane 1) and an uncloned SCp2–Id-1-transfected pooled population (lane 2). Cells were cultured in serum-free medium. (D) Gelatin zymogram of SCp2–Id-1A (lanes 1, 3, and 5) and SCp2–Id-1E (lanes 2, 4, and 6) cell-conditioned media incubated with <t>DMSO</t> (lanes 1 and 2), the MMP inhibitor GM6001 (0.2 mM in DMSO) (lanes 3 and 4), or the serine proteinase inhibitor <t>PMSF</t> (5 mM in DMSO) (lanes 5 and 6). (E) Casein zymogram of conditioned media from SCp2–Id-1A (lanes 1 and 3) and SCp2–Id-1E (lanes 2 and 4) cells incubated with DMSO (lanes 1 and 2) or GM6001 (lanes 3 and 4). In panels A through D, arrows mark the positions of the 120-kDa MMP.
    Pmsf, 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
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    Image Search Results


    Inhibitory effects of TA on HAT activity. (A) A colorimetric assay was performed to measure HAT activity in the presence of TA at the indicated concentrations. The results are presented as percentages relative to the control sample, which was incubated without TA (left panel). The IC 50 value of TA inhibition of HAT activity was calculated using Prism software (right panel). (B) An in vitro HAT assay using autoradiography was performed to evaluate the effect of the anti-HAT activity of TA on the acetylation of a synthetic H4 peptide by a HeLa NE. The arrow indicates histone H4 (left panel). The autoradiography was quantified using Image J software (right panel). The values presented are the means ± SD of two independent experiments. Means with different superscript letters are significantly different, p

    Journal: Molecular Metabolism

    Article Title: Tannic acid, a novel histone acetyltransferase inhibitor, prevents non-alcoholic fatty liver disease both in vivo and in vitro model

    doi: 10.1016/j.molmet.2018.11.001

    Figure Lengend Snippet: Inhibitory effects of TA on HAT activity. (A) A colorimetric assay was performed to measure HAT activity in the presence of TA at the indicated concentrations. The results are presented as percentages relative to the control sample, which was incubated without TA (left panel). The IC 50 value of TA inhibition of HAT activity was calculated using Prism software (right panel). (B) An in vitro HAT assay using autoradiography was performed to evaluate the effect of the anti-HAT activity of TA on the acetylation of a synthetic H4 peptide by a HeLa NE. The arrow indicates histone H4 (left panel). The autoradiography was quantified using Image J software (right panel). The values presented are the means ± SD of two independent experiments. Means with different superscript letters are significantly different, p

    Article Snippet: For autoradiography-based in vitro HAT activity assays, HeLa cell nuclear extract was incubated with HAT assay buffer (50 mM HEPES, pH 8.0; 10% glycerol; 1 mM DTT; 1 mM phenylmethylsulfonyl fluoride; and 10 mM sodium butyrate), 1 μL [3 H] acetyl-CoA, and 5 μg of biotinylated-H4 peptide (Millipore, Billerica, MA, USA) along with TA at the indicated concentrations at 30 °C for 1 h. The reactions were stopped by adding 5 × sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS–PAGE) sample buffer and samples were separated on 15% SDS–PAGE gels, followed by autoradiographic analysis.

    Techniques: HAT Assay, Activity Assay, Colorimetric Assay, Incubation, Inhibition, Software, In Vitro, Autoradiography

    MMP inhibitors prevent the increases in vascular gelatinolytic activity, in ROS concentrations, and in lucigenin chemiluminescence after intraluminal incubation of rabbit aorta with MMP-2. (A) Representative photomicrographs of aortic in situ zymography experiments showing that MMP-2 gelatinolytic activity was totally inhibited by phenanthroline (Phe, 100 µM) or doxycycline (Doxy, 100 µM), but not by phenylmethylsulfonyl fluoride (PMSF, 100 µM). The arteries were incubated with the inhibitors together with MMP-2 (16 nM) intraluminally for 30 min. (B) Quantification of aortic gelatinolytic activity measured in experiments described in A. (C) Representative photomicrographs of aortic sections incubated with DHE. Red fluorescence reflects ROS production and shows that incubation with MMP-2 (16 nM) intraluminally for 30 min increased ROS production, and this effect was totally inhibited by phenanthroline (Phe, 100 µM) or doxycycline (Doxy, 100 µM), but not by phenylmethylsulfonyl fluoride (PMSF, 100 µM). The experiments were carried out as described in A. (D), Quantification of aortic fluorescence of DHE measured in experiments described in C. (E), Quantification of lucigenin chemiluminescence measured in fresh aortas after intraluminal incubation with MMP-2 (16 nM) or vehicle for 30 min in absence or presence of MMP inhibitors, doxycycline (Doxy 100 µM) or GM6001 (10 µM). Data are shown as the mean ± SEM (n = 4–5/group). * P

    Journal: Redox Biology

    Article Title: Matrix metalloproteinase-2-induced epidermal growth factor receptor transactivation impairs redox balance in vascular smooth muscle cells and facilitates vascular contraction

    doi: 10.1016/j.redox.2018.07.005

    Figure Lengend Snippet: MMP inhibitors prevent the increases in vascular gelatinolytic activity, in ROS concentrations, and in lucigenin chemiluminescence after intraluminal incubation of rabbit aorta with MMP-2. (A) Representative photomicrographs of aortic in situ zymography experiments showing that MMP-2 gelatinolytic activity was totally inhibited by phenanthroline (Phe, 100 µM) or doxycycline (Doxy, 100 µM), but not by phenylmethylsulfonyl fluoride (PMSF, 100 µM). The arteries were incubated with the inhibitors together with MMP-2 (16 nM) intraluminally for 30 min. (B) Quantification of aortic gelatinolytic activity measured in experiments described in A. (C) Representative photomicrographs of aortic sections incubated with DHE. Red fluorescence reflects ROS production and shows that incubation with MMP-2 (16 nM) intraluminally for 30 min increased ROS production, and this effect was totally inhibited by phenanthroline (Phe, 100 µM) or doxycycline (Doxy, 100 µM), but not by phenylmethylsulfonyl fluoride (PMSF, 100 µM). The experiments were carried out as described in A. (D), Quantification of aortic fluorescence of DHE measured in experiments described in C. (E), Quantification of lucigenin chemiluminescence measured in fresh aortas after intraluminal incubation with MMP-2 (16 nM) or vehicle for 30 min in absence or presence of MMP inhibitors, doxycycline (Doxy 100 µM) or GM6001 (10 µM). Data are shown as the mean ± SEM (n = 4–5/group). * P

    Article Snippet: Tyrphostin AG 1478, Phenylephrine, Apocynin, Peg-Catalase (PG-Cat), Dihydroethidium (DHE), phenanthroline, Phenylmethylsulfonyl fluoride were purchased from Sigma Chemical Co. (St. Louis, MO, USA).

    Techniques: Activity Assay, Incubation, In Situ, Zymography, Fluorescence

    Expression of a 120-kDa gelatinase by Id-1-expressing mammary epithelial cells. (A) Gelatin zymogram of conditioned media from SCp2–Id-1A (lane 1) and SCp2–Id-1E (lane 2) cells. Cells were cultured in serum-free medium, and conditioned media were harvested and analyzed on a gelatin substrate gel, as described in Materials and Methods. (B) Gelatin zymogram of conditioned media from SCp2–Id-1A cells either growth arrested by serum deprivation (lane 1) or growing in 5% serum (lane 2). (C) Gelatin zymogram of conditioned media from control SCp2 cells (lane 1) and an uncloned SCp2–Id-1-transfected pooled population (lane 2). Cells were cultured in serum-free medium. (D) Gelatin zymogram of SCp2–Id-1A (lanes 1, 3, and 5) and SCp2–Id-1E (lanes 2, 4, and 6) cell-conditioned media incubated with DMSO (lanes 1 and 2), the MMP inhibitor GM6001 (0.2 mM in DMSO) (lanes 3 and 4), or the serine proteinase inhibitor PMSF (5 mM in DMSO) (lanes 5 and 6). (E) Casein zymogram of conditioned media from SCp2–Id-1A (lanes 1 and 3) and SCp2–Id-1E (lanes 2 and 4) cells incubated with DMSO (lanes 1 and 2) or GM6001 (lanes 3 and 4). In panels A through D, arrows mark the positions of the 120-kDa MMP.

    Journal: Molecular and Cellular Biology

    Article Title: A Novel Pathway for Mammary Epithelial Cell Invasion Induced by the Helix-Loop-Helix Protein Id-1

    doi:

    Figure Lengend Snippet: Expression of a 120-kDa gelatinase by Id-1-expressing mammary epithelial cells. (A) Gelatin zymogram of conditioned media from SCp2–Id-1A (lane 1) and SCp2–Id-1E (lane 2) cells. Cells were cultured in serum-free medium, and conditioned media were harvested and analyzed on a gelatin substrate gel, as described in Materials and Methods. (B) Gelatin zymogram of conditioned media from SCp2–Id-1A cells either growth arrested by serum deprivation (lane 1) or growing in 5% serum (lane 2). (C) Gelatin zymogram of conditioned media from control SCp2 cells (lane 1) and an uncloned SCp2–Id-1-transfected pooled population (lane 2). Cells were cultured in serum-free medium. (D) Gelatin zymogram of SCp2–Id-1A (lanes 1, 3, and 5) and SCp2–Id-1E (lanes 2, 4, and 6) cell-conditioned media incubated with DMSO (lanes 1 and 2), the MMP inhibitor GM6001 (0.2 mM in DMSO) (lanes 3 and 4), or the serine proteinase inhibitor PMSF (5 mM in DMSO) (lanes 5 and 6). (E) Casein zymogram of conditioned media from SCp2–Id-1A (lanes 1 and 3) and SCp2–Id-1E (lanes 2 and 4) cells incubated with DMSO (lanes 1 and 2) or GM6001 (lanes 3 and 4). In panels A through D, arrows mark the positions of the 120-kDa MMP.

    Article Snippet: Concentrated conditioned medium was mixed with nonreducing Laemmli sample buffer and incubated at 37°C for 15 min. After electrophoresis, the gels were incubated for 1 h in 2.5% Triton X-100 at room temperature, followed by 24 to 48 h in substrate buffer (100 mM Tris-HCl [pH 7.4]–15 mM CaCl2 ) in the absence or presence of GM6001 (0.2 mM in DMSO; supplied by Glycomed Corporation and obtained from Z. Werb [ ]), EDTA (10 mM), ortho -phenanthroline (1 mM in DMSO; Sigma), PMSF (5 mM), or AEBSF (0.5 mM; Calbiochem).

    Techniques: Expressing, Cell Culture, Transfection, Incubation

    The cortactin repeat has a dynamic conformation in solution (A) The endogenous cysteine residues 112 and 246 of the cortactin repeat were crosslinked and the ability of the crosslinked protein to bind F-actin was tested in a co-sedimentation assay (Materials and Methods). The pellet (P) and supernatant (S) were analyzed on 12% SDS-PAGE. Lanes 1 and 2, uncrosslinked cortactin; lanes 3 and 4, disulfide crosslinked cortactin (xl-cortactin); lanes 5 and 6, reduction of the disulfide crosslink with 10mM DTT. (B) The ability of the NEM-labeled cortactin repeat (at residues C112 and C246) to bind to F-actin was assessed using a co-sedimentation assay (Materials and Methods). The pellet and supernatant were analyzed on 12% SDS-PAGE. Lanes 1 and 2, non-labeled cortactin; lanes 3 and 4, NEM-labeled cortactin. (C) Crosslinking of cortactin repeat constructs containing pairs of cysteine residues at positions 83 and 306 (left), 83 and 246 (middle), and 83 and 112 (right). Prior to each crosslinking reaction, DTT was removed from the samples on a Sephadex G-50 spin column equilibrated with 20 mM HEPES pH 7.0, 0.1 M KCl, and 100μM PMSF. The crosslinking reactions were performed on ice and catalyzed by addition of 10μM CuSO 4 . Each cysteine pair was crosslinked either directly by disulfide bond formation (zero length crosslink), or using the crosslinking reagents DBB (4.4 Å span) or MTS-6 (9.6 Å span).

    Journal: Cell motility and the cytoskeleton

    Article Title: The Actin-binding Domain of Cortactin is Dynamic and Unstructured and Affects Lateral and Longitudinal Contacts in F-actin

    doi: 10.1002/cm.20328

    Figure Lengend Snippet: The cortactin repeat has a dynamic conformation in solution (A) The endogenous cysteine residues 112 and 246 of the cortactin repeat were crosslinked and the ability of the crosslinked protein to bind F-actin was tested in a co-sedimentation assay (Materials and Methods). The pellet (P) and supernatant (S) were analyzed on 12% SDS-PAGE. Lanes 1 and 2, uncrosslinked cortactin; lanes 3 and 4, disulfide crosslinked cortactin (xl-cortactin); lanes 5 and 6, reduction of the disulfide crosslink with 10mM DTT. (B) The ability of the NEM-labeled cortactin repeat (at residues C112 and C246) to bind to F-actin was assessed using a co-sedimentation assay (Materials and Methods). The pellet and supernatant were analyzed on 12% SDS-PAGE. Lanes 1 and 2, non-labeled cortactin; lanes 3 and 4, NEM-labeled cortactin. (C) Crosslinking of cortactin repeat constructs containing pairs of cysteine residues at positions 83 and 306 (left), 83 and 246 (middle), and 83 and 112 (right). Prior to each crosslinking reaction, DTT was removed from the samples on a Sephadex G-50 spin column equilibrated with 20 mM HEPES pH 7.0, 0.1 M KCl, and 100μM PMSF. The crosslinking reactions were performed on ice and catalyzed by addition of 10μM CuSO 4 . Each cysteine pair was crosslinked either directly by disulfide bond formation (zero length crosslink), or using the crosslinking reagents DBB (4.4 Å span) or MTS-6 (9.6 Å span).

    Article Snippet: Sephadex G-50, N-ethylmaleimide (NEM), ATP, phalloidin, DTT and PMSF were purchased from Sigma Chemical Company (St. Louis, MO).

    Techniques: Sedimentation, SDS Page, Labeling, Construct