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  • 99
    Millipore pronase
    Pronase, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 5494 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Merck KGaA pronase e
    Cultures of hepatic stellate cells isolated via iohexol density gradient centrifugation without or with subsequent cell sorting. Hepatic stellate cells (HSC) were isolated from 40-week-old C57BL/6J mice using enzymatic digestion of the liver based on <t>pronase</t> and collagenase, followed by density gradient centrifugation in 8% iohexol. Cells were cultured directly after the gradient for one day (a) and four days (b), where Kupffer cells (indicated by arrows) can be found in the HSC culture. Highly pure HSC after additional fluorescence-activated cell sorting (FACS) after one (c) and four days of culture are shown (d). Expression analysis of desmin (e) and phalloidin (f) of HSC after four days of culture, indicating proper maturation of HSC.
    Pronase E, supplied by Merck KGaA, used in various techniques. Bioz Stars score: 93/100, based on 272 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Boehringer Mannheim pronase
    SDS-PAGE analysis (with sample boiling and urea) of <t>pronase-treated</t> 125 I-CPE bound to BBMs at RT. 125 I-CPE was bound to BBMs at RT, and after removal of unbound 125 I-CPE, samples were treated for 60 min at 4°C with 300 μg of pronase (lane 2) or subjected to extraction with 1% Triton X-100 and then treated for 60 min at 4°C with 300 μg of pronase (lane 3). Lane 1 shows a similarly prepared control sample that was kept at 4°C for 60 min without pronase treatment. The samples shown in lanes 1 and 2 were washed with cold DPBS containing protease inhibitors (see Materials and Methods) after pronase treatment, but before Triton X-100 extraction, while cold DPBS containing similar protease inhibitors was added to the lane 3 sample after the conclusion of the pronase treatment. Each sample was then electrophoresed on preparative SDS–6% polyacrylamide gels without sample boiling or β-mercaptoethanol treatment. Slices of these preparative gels corresponding to large complex were then excised, and proteins in these gel slices were electroeluted, concentrated, treated with urea, boiled, and electrophoresed on an SDS–15% polyacrylamide gel containing urea. Migrations of molecular mass markers are shown at the right. Migration of 125 I-CPE is shown at the left. The gel shown is representative of gels used in five independent repetitions of the experiment.
    Pronase, supplied by Boehringer Mannheim, used in various techniques. Bioz Stars score: 93/100, based on 387 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pronase/product/Boehringer Mannheim
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    99
    Millipore streptomyces griseus
    SDS-PAGE analysis (with sample boiling and urea) of <t>pronase-treated</t> 125 I-CPE bound to BBMs at RT. 125 I-CPE was bound to BBMs at RT, and after removal of unbound 125 I-CPE, samples were treated for 60 min at 4°C with 300 μg of pronase (lane 2) or subjected to extraction with 1% Triton X-100 and then treated for 60 min at 4°C with 300 μg of pronase (lane 3). Lane 1 shows a similarly prepared control sample that was kept at 4°C for 60 min without pronase treatment. The samples shown in lanes 1 and 2 were washed with cold DPBS containing protease inhibitors (see Materials and Methods) after pronase treatment, but before Triton X-100 extraction, while cold DPBS containing similar protease inhibitors was added to the lane 3 sample after the conclusion of the pronase treatment. Each sample was then electrophoresed on preparative SDS–6% polyacrylamide gels without sample boiling or β-mercaptoethanol treatment. Slices of these preparative gels corresponding to large complex were then excised, and proteins in these gel slices were electroeluted, concentrated, treated with urea, boiled, and electrophoresed on an SDS–15% polyacrylamide gel containing urea. Migrations of molecular mass markers are shown at the right. Migration of 125 I-CPE is shown at the left. The gel shown is representative of gels used in five independent repetitions of the experiment.
    Streptomyces Griseus, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1501 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Kaken Pharmaceutical pronase
    Figure 3. Lung section from a 2 cm-thick specimen stored in 10% neutral buffered formalin for 22 mo. Immunoperoxidase SAB-staining, obtained with anti-BHV-1 serum, <t>pronase-pretreated</t> for 30 min. Bar = 100 μm.
    Pronase, supplied by Kaken Pharmaceutical, used in various techniques. Bioz Stars score: 92/100, based on 93 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore protease from streptomyces griseus
    Figure 3. Lung section from a 2 cm-thick specimen stored in 10% neutral buffered formalin for 22 mo. Immunoperoxidase SAB-staining, obtained with anti-BHV-1 serum, <t>pronase-pretreated</t> for 30 min. Bar = 100 μm.
    Protease From Streptomyces Griseus, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 102 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    97
    Thermo Fisher pronase
    Figure 3. Lung section from a 2 cm-thick specimen stored in 10% neutral buffered formalin for 22 mo. Immunoperoxidase SAB-staining, obtained with anti-BHV-1 serum, <t>pronase-pretreated</t> for 30 min. Bar = 100 μm.
    Pronase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 97/100, based on 251 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Agilent technologies pronase
    Figure 3. Lung section from a 2 cm-thick specimen stored in 10% neutral buffered formalin for 22 mo. Immunoperoxidase SAB-staining, obtained with anti-BHV-1 serum, <t>pronase-pretreated</t> for 30 min. Bar = 100 μm.
    Pronase, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 92/100, based on 121 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Merck & Co pronase
    Inhibitory activities on B. thailandensis E264 motility on swarm agar of cell-free supernatant of B. pseudomallei 1710a after filtration, protein digestion, or incubation under different conditions. (A) Inhibition zone in retentate and filtrate fractions following filtration through membranes with different molecular weight cut-off sizes. (B) Inhibitory activity of cell-free supernatant of B. pseudomallei 1710a after treatments with proteinase K (left panel) and <t>pronase</t> (right panel), (a) no treatment control, (b) treatment with proteinase K or pronase, (c) Enzymes alone (no supernatants) were spotted onto the agar in the same concentration as a control. (C) Inhibition activity of cell-free supernatant of B. pseudomallei 1710a following incubation at different pH at 37°C for 24 h. (D) Inhibition activity of cell-free supernatant of B. pseudomallei 1710a following incubation at different temperatures for 24 h. (E) Inhibition activity of cell-free supernatant of B. pseudomallei 1710a following incubation at different salt concentrations at 37°C for 24 h. The controls for (A) , (C) , (D) and (E) are B. thailandensis E264 swarming without the inoculation of cell-free supernatant. Plates were incubated at 37°C for 24 h.
    Pronase, supplied by Merck & Co, used in various techniques. Bioz Stars score: 93/100, based on 64 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Fisher Scientific pronase
    Inhibitory activities on B. thailandensis E264 motility on swarm agar of cell-free supernatant of B. pseudomallei 1710a after filtration, protein digestion, or incubation under different conditions. (A) Inhibition zone in retentate and filtrate fractions following filtration through membranes with different molecular weight cut-off sizes. (B) Inhibitory activity of cell-free supernatant of B. pseudomallei 1710a after treatments with proteinase K (left panel) and <t>pronase</t> (right panel), (a) no treatment control, (b) treatment with proteinase K or pronase, (c) Enzymes alone (no supernatants) were spotted onto the agar in the same concentration as a control. (C) Inhibition activity of cell-free supernatant of B. pseudomallei 1710a following incubation at different pH at 37°C for 24 h. (D) Inhibition activity of cell-free supernatant of B. pseudomallei 1710a following incubation at different temperatures for 24 h. (E) Inhibition activity of cell-free supernatant of B. pseudomallei 1710a following incubation at different salt concentrations at 37°C for 24 h. The controls for (A) , (C) , (D) and (E) are B. thailandensis E264 swarming without the inoculation of cell-free supernatant. Plates were incubated at 37°C for 24 h.
    Pronase, supplied by Fisher Scientific, used in various techniques. Bioz Stars score: 92/100, based on 63 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Promega pronase
    Identification of Q24DA secondary targets by DARTS assay. Cell lysates from C7258 were incubated with Q24DA, an inactive structural analog (SR27911), or DMSO (vehicle control); digested with <t>pronase;</t> and subjected to two-dimensional gel electrophoresis
    Pronase, supplied by Promega, used in various techniques. Bioz Stars score: 92/100, based on 31 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Avantor pronase
    Identification of Q24DA secondary targets by DARTS assay. Cell lysates from C7258 were incubated with Q24DA, an inactive structural analog (SR27911), or DMSO (vehicle control); digested with <t>pronase;</t> and subjected to two-dimensional gel electrophoresis
    Pronase, supplied by Avantor, used in various techniques. Bioz Stars score: 91/100, based on 25 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    SERVA Electrophoresis pronase
    Identification of Q24DA secondary targets by DARTS assay. Cell lysates from C7258 were incubated with Q24DA, an inactive structural analog (SR27911), or DMSO (vehicle control); digested with <t>pronase;</t> and subjected to two-dimensional gel electrophoresis
    Pronase, supplied by SERVA Electrophoresis, used in various techniques. Bioz Stars score: 92/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Cultures of hepatic stellate cells isolated via iohexol density gradient centrifugation without or with subsequent cell sorting. Hepatic stellate cells (HSC) were isolated from 40-week-old C57BL/6J mice using enzymatic digestion of the liver based on pronase and collagenase, followed by density gradient centrifugation in 8% iohexol. Cells were cultured directly after the gradient for one day (a) and four days (b), where Kupffer cells (indicated by arrows) can be found in the HSC culture. Highly pure HSC after additional fluorescence-activated cell sorting (FACS) after one (c) and four days of culture are shown (d). Expression analysis of desmin (e) and phalloidin (f) of HSC after four days of culture, indicating proper maturation of HSC.

    Journal: Analytical cellular pathology (Amsterdam)

    Article Title: Isolation and Time Lapse Microscopy of Highly Pure Hepatic Stellate Cells

    doi: 10.1155/2015/417023

    Figure Lengend Snippet: Cultures of hepatic stellate cells isolated via iohexol density gradient centrifugation without or with subsequent cell sorting. Hepatic stellate cells (HSC) were isolated from 40-week-old C57BL/6J mice using enzymatic digestion of the liver based on pronase and collagenase, followed by density gradient centrifugation in 8% iohexol. Cells were cultured directly after the gradient for one day (a) and four days (b), where Kupffer cells (indicated by arrows) can be found in the HSC culture. Highly pure HSC after additional fluorescence-activated cell sorting (FACS) after one (c) and four days of culture are shown (d). Expression analysis of desmin (e) and phalloidin (f) of HSC after four days of culture, indicating proper maturation of HSC.

    Article Snippet: Second, perfusion buffer 2 (8 g/L NaCl, 400 mg/L KCl, 78 mg/L NaH2 PO4 × H2 O, 151 mg/L NaHPO4 × 2 H2 O, 2380 mg/L HEPES, 350 mg/L NaHCO3 , 560 mg/L CaCl2 × 2 H2 O, and 6 mg/L phenol red, adjusted to pH 7.3–7.4 using 10 N NaOH, sterile filtered, and kept at 4°C until use) was applied for 4.5 minutes and supplemented with 0.5 mg/mL pronase E (Merck, Darmstadt, Germany).

    Techniques: Isolation, Gradient Centrifugation, FACS, Mouse Assay, Cell Culture, Fluorescence, Expressing

    Optimization of the isolation of primary hepatic stellate cells (HSC) based on iohexol density gradient centrifugation and fluorescence-activated cell sorting (FACS) (schematic depiction). In both strategies for cell purification, the mouse is anaesthetized before surgery, and the liver is then perfused via the Vena portae and drained through the Vena cava inferior using a two-step perfusion of the enzymes pronase and collagenase (a). Liver cells are harvested by gently tearing the liver into bits (b), followed by a postdigestion using a combination of both enzymes (c). The liver cells are subjected to iohexol density gradient centrifugation, after which HSC and Kupffer cells are located in the interphase between iohexol and buffer (d). The enriched HSC layer containing HSC, HSC-Kupffer cell doublets, and cellular debris can be used directly for cell culture of HSC (e) or can be cleared from HSC-Kupffer cell doublets and cellular debris using FACS based on the autofluorescence of retinol, using the UV laser of the cell sorter, resulting in highly pure HSC (f).

    Journal: Analytical cellular pathology (Amsterdam)

    Article Title: Isolation and Time Lapse Microscopy of Highly Pure Hepatic Stellate Cells

    doi: 10.1155/2015/417023

    Figure Lengend Snippet: Optimization of the isolation of primary hepatic stellate cells (HSC) based on iohexol density gradient centrifugation and fluorescence-activated cell sorting (FACS) (schematic depiction). In both strategies for cell purification, the mouse is anaesthetized before surgery, and the liver is then perfused via the Vena portae and drained through the Vena cava inferior using a two-step perfusion of the enzymes pronase and collagenase (a). Liver cells are harvested by gently tearing the liver into bits (b), followed by a postdigestion using a combination of both enzymes (c). The liver cells are subjected to iohexol density gradient centrifugation, after which HSC and Kupffer cells are located in the interphase between iohexol and buffer (d). The enriched HSC layer containing HSC, HSC-Kupffer cell doublets, and cellular debris can be used directly for cell culture of HSC (e) or can be cleared from HSC-Kupffer cell doublets and cellular debris using FACS based on the autofluorescence of retinol, using the UV laser of the cell sorter, resulting in highly pure HSC (f).

    Article Snippet: Second, perfusion buffer 2 (8 g/L NaCl, 400 mg/L KCl, 78 mg/L NaH2 PO4 × H2 O, 151 mg/L NaHPO4 × 2 H2 O, 2380 mg/L HEPES, 350 mg/L NaHCO3 , 560 mg/L CaCl2 × 2 H2 O, and 6 mg/L phenol red, adjusted to pH 7.3–7.4 using 10 N NaOH, sterile filtered, and kept at 4°C until use) was applied for 4.5 minutes and supplemented with 0.5 mg/mL pronase E (Merck, Darmstadt, Germany).

    Techniques: Isolation, Gradient Centrifugation, Fluorescence, FACS, Purification, Cell Culture

    Hepatic stellate cell functionality in vitro . Hepatic stellate cells (HSC) were isolated from 40-week-old C57BL/6J mice using enzymatic digestion of the liver based on pronase and collagenase, followed by density gradient centrifugation in 8% iohexol and fluorescence-activated cell sorting (FACS). The HSC were cultured in DMEM with 10% fetal calf serum, and some plates were stimulated with lipopolysaccharides (LPS) at 100 ng/mL (after five days of culture) for another 48 hours. Changes in the cell number during culture were determined from time lapse microscopy (a) and statistical summary (b). HSC were cultured for five days on tissue culture-treated polystyrene in DMEM with 10% fetal calf serum including culture inserts for self-insertion (“scratch assay”). To start horizontal migration, the plastic inserts were removed and HSC migrated (c) and were quantified using software (d). HSC were cultured for designated periods and quantitative real-time PCR was performed to study the expression of α smooth muscle actin ( α SMA), collagen 1 (Col1A1), or the transforming growth factor β (TGF β ) as markers of HSC activation. Gene expression was normalized to β -actin expression of cells that were lysed directly after isolation at day zero (e). Mean ± SD of three independent experiments.

    Journal: Analytical cellular pathology (Amsterdam)

    Article Title: Isolation and Time Lapse Microscopy of Highly Pure Hepatic Stellate Cells

    doi: 10.1155/2015/417023

    Figure Lengend Snippet: Hepatic stellate cell functionality in vitro . Hepatic stellate cells (HSC) were isolated from 40-week-old C57BL/6J mice using enzymatic digestion of the liver based on pronase and collagenase, followed by density gradient centrifugation in 8% iohexol and fluorescence-activated cell sorting (FACS). The HSC were cultured in DMEM with 10% fetal calf serum, and some plates were stimulated with lipopolysaccharides (LPS) at 100 ng/mL (after five days of culture) for another 48 hours. Changes in the cell number during culture were determined from time lapse microscopy (a) and statistical summary (b). HSC were cultured for five days on tissue culture-treated polystyrene in DMEM with 10% fetal calf serum including culture inserts for self-insertion (“scratch assay”). To start horizontal migration, the plastic inserts were removed and HSC migrated (c) and were quantified using software (d). HSC were cultured for designated periods and quantitative real-time PCR was performed to study the expression of α smooth muscle actin ( α SMA), collagen 1 (Col1A1), or the transforming growth factor β (TGF β ) as markers of HSC activation. Gene expression was normalized to β -actin expression of cells that were lysed directly after isolation at day zero (e). Mean ± SD of three independent experiments.

    Article Snippet: Second, perfusion buffer 2 (8 g/L NaCl, 400 mg/L KCl, 78 mg/L NaH2 PO4 × H2 O, 151 mg/L NaHPO4 × 2 H2 O, 2380 mg/L HEPES, 350 mg/L NaHCO3 , 560 mg/L CaCl2 × 2 H2 O, and 6 mg/L phenol red, adjusted to pH 7.3–7.4 using 10 N NaOH, sterile filtered, and kept at 4°C until use) was applied for 4.5 minutes and supplemented with 0.5 mg/mL pronase E (Merck, Darmstadt, Germany).

    Techniques: In Vitro, Isolation, Mouse Assay, Gradient Centrifugation, Fluorescence, FACS, Cell Culture, Time-lapse Microscopy, Migration, Software, Real-time Polymerase Chain Reaction, Expressing, Activation Assay

    SDS-PAGE analysis (with sample boiling and urea) of pronase-treated 125 I-CPE bound to BBMs at RT. 125 I-CPE was bound to BBMs at RT, and after removal of unbound 125 I-CPE, samples were treated for 60 min at 4°C with 300 μg of pronase (lane 2) or subjected to extraction with 1% Triton X-100 and then treated for 60 min at 4°C with 300 μg of pronase (lane 3). Lane 1 shows a similarly prepared control sample that was kept at 4°C for 60 min without pronase treatment. The samples shown in lanes 1 and 2 were washed with cold DPBS containing protease inhibitors (see Materials and Methods) after pronase treatment, but before Triton X-100 extraction, while cold DPBS containing similar protease inhibitors was added to the lane 3 sample after the conclusion of the pronase treatment. Each sample was then electrophoresed on preparative SDS–6% polyacrylamide gels without sample boiling or β-mercaptoethanol treatment. Slices of these preparative gels corresponding to large complex were then excised, and proteins in these gel slices were electroeluted, concentrated, treated with urea, boiled, and electrophoresed on an SDS–15% polyacrylamide gel containing urea. Migrations of molecular mass markers are shown at the right. Migration of 125 I-CPE is shown at the left. The gel shown is representative of gels used in five independent repetitions of the experiment.

    Journal: Infection and Immunity

    Article Title: Characterization of Membrane-Associated Clostridium perfringens Enterotoxin following Pronase Treatment

    doi:

    Figure Lengend Snippet: SDS-PAGE analysis (with sample boiling and urea) of pronase-treated 125 I-CPE bound to BBMs at RT. 125 I-CPE was bound to BBMs at RT, and after removal of unbound 125 I-CPE, samples were treated for 60 min at 4°C with 300 μg of pronase (lane 2) or subjected to extraction with 1% Triton X-100 and then treated for 60 min at 4°C with 300 μg of pronase (lane 3). Lane 1 shows a similarly prepared control sample that was kept at 4°C for 60 min without pronase treatment. The samples shown in lanes 1 and 2 were washed with cold DPBS containing protease inhibitors (see Materials and Methods) after pronase treatment, but before Triton X-100 extraction, while cold DPBS containing similar protease inhibitors was added to the lane 3 sample after the conclusion of the pronase treatment. Each sample was then electrophoresed on preparative SDS–6% polyacrylamide gels without sample boiling or β-mercaptoethanol treatment. Slices of these preparative gels corresponding to large complex were then excised, and proteins in these gel slices were electroeluted, concentrated, treated with urea, boiled, and electrophoresed on an SDS–15% polyacrylamide gel containing urea. Migrations of molecular mass markers are shown at the right. Migration of 125 I-CPE is shown at the left. The gel shown is representative of gels used in five independent repetitions of the experiment.

    Article Snippet: Pronase was purchased from Boehringer Mannheim; antipain, chymostatin, leupeptin, pepstatin, phenylmethylsulfonyl fluoride, dimethyl sulfoxide, EDTA, and Azocoll (Azo dye-impregnated collagen) were purchased from Sigma; and AEBSF [4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride] was obtained from ICN Biomedicals.

    Techniques: SDS Page, Migration

    SDS-PAGE analysis of pronase-treated 125 I-CPE bound to BBMs at 4°C. 125 I-CPE was bound to BBMs at 4°C, and after removal of unbound 125 I-CPE, these samples were either treated for 60 min at 4°C with 300 μg of pronase (lane 1), kept for 60 min at 4°C as an untreated control (lane 2), or subjected to extraction with cold DPBS containing 1% Triton X-100 before treatment for 60 min at 4°C with 300 μg of pronase (lane 3). The lane 1 and 2 samples were washed with cold DPBS containing protease inhibitors (see Materials and Methods) before being subjected to extraction with 1% Triton X-100, while cold DPBS containing similar protease inhibitors were added to the lane 3 sample at the completion of pronase treatment. Each sample was then electrophoresed on preparative native 6% polyacrylamide gel, and samples containing small complex were excised from these gels. Proteins in the excised gel slices were electroeluted, concentrated, treated with urea, boiled, and analyzed on an SDS–12% polyacrylamide gel containing urea. Note that in lane 2 only half the volume of sample was loaded compared to that loaded in lanes 1 and 3. Migrations of molecular mass markers are shown at the right, while the migration of free 125 I-CPE is shown at the left. The gel shown is representative of gels used in five independent repetitions of the experiment. Note that the presence of small amounts of intact 125 I-CPE in lanes 1 and 3 is not due to spillover, since similar results were observed when these samples were each run on individual gels.

    Journal: Infection and Immunity

    Article Title: Characterization of Membrane-Associated Clostridium perfringens Enterotoxin following Pronase Treatment

    doi:

    Figure Lengend Snippet: SDS-PAGE analysis of pronase-treated 125 I-CPE bound to BBMs at 4°C. 125 I-CPE was bound to BBMs at 4°C, and after removal of unbound 125 I-CPE, these samples were either treated for 60 min at 4°C with 300 μg of pronase (lane 1), kept for 60 min at 4°C as an untreated control (lane 2), or subjected to extraction with cold DPBS containing 1% Triton X-100 before treatment for 60 min at 4°C with 300 μg of pronase (lane 3). The lane 1 and 2 samples were washed with cold DPBS containing protease inhibitors (see Materials and Methods) before being subjected to extraction with 1% Triton X-100, while cold DPBS containing similar protease inhibitors were added to the lane 3 sample at the completion of pronase treatment. Each sample was then electrophoresed on preparative native 6% polyacrylamide gel, and samples containing small complex were excised from these gels. Proteins in the excised gel slices were electroeluted, concentrated, treated with urea, boiled, and analyzed on an SDS–12% polyacrylamide gel containing urea. Note that in lane 2 only half the volume of sample was loaded compared to that loaded in lanes 1 and 3. Migrations of molecular mass markers are shown at the right, while the migration of free 125 I-CPE is shown at the left. The gel shown is representative of gels used in five independent repetitions of the experiment. Note that the presence of small amounts of intact 125 I-CPE in lanes 1 and 3 is not due to spillover, since similar results were observed when these samples were each run on individual gels.

    Article Snippet: Pronase was purchased from Boehringer Mannheim; antipain, chymostatin, leupeptin, pepstatin, phenylmethylsulfonyl fluoride, dimethyl sulfoxide, EDTA, and Azocoll (Azo dye-impregnated collagen) were purchased from Sigma; and AEBSF [4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride] was obtained from ICN Biomedicals.

    Techniques: SDS Page, Migration

    Effects of washing BBMs on pronase-induced release of specifically bound 125 I-CPE. 125 I-CPE was bound to BBMs at 4°C (A) or at RT (B) in the presence or absence of excess unlabeled CPE. After unbound 125 I-CPE was removed, the BBMs were incubated at 4°C with or without pronase (300 μg) for 5 min to 1 h. Circles, specifically bound radioactivity remaining in these pronase-treated BBMs; squares, specifically bound radioactivity remaining in the non-pronase-treated BBMs. After this pronase treatment, BBMs were pelleted and then their radioactivity was either directly counted in a gamma counter (nonwashed samples [open symbols]) or counted after the pellets were washed three times with cold DPBS containing protease inhibitors (see the text) (washed samples [filled symbols]). Values are means ± standard errors of the means. Error bars not shown indicate that the values were too small to depict. Each experiment was repeated three times with triplicate samples.

    Journal: Infection and Immunity

    Article Title: Characterization of Membrane-Associated Clostridium perfringens Enterotoxin following Pronase Treatment

    doi:

    Figure Lengend Snippet: Effects of washing BBMs on pronase-induced release of specifically bound 125 I-CPE. 125 I-CPE was bound to BBMs at 4°C (A) or at RT (B) in the presence or absence of excess unlabeled CPE. After unbound 125 I-CPE was removed, the BBMs were incubated at 4°C with or without pronase (300 μg) for 5 min to 1 h. Circles, specifically bound radioactivity remaining in these pronase-treated BBMs; squares, specifically bound radioactivity remaining in the non-pronase-treated BBMs. After this pronase treatment, BBMs were pelleted and then their radioactivity was either directly counted in a gamma counter (nonwashed samples [open symbols]) or counted after the pellets were washed three times with cold DPBS containing protease inhibitors (see the text) (washed samples [filled symbols]). Values are means ± standard errors of the means. Error bars not shown indicate that the values were too small to depict. Each experiment was repeated three times with triplicate samples.

    Article Snippet: Pronase was purchased from Boehringer Mannheim; antipain, chymostatin, leupeptin, pepstatin, phenylmethylsulfonyl fluoride, dimethyl sulfoxide, EDTA, and Azocoll (Azo dye-impregnated collagen) were purchased from Sigma; and AEBSF [4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride] was obtained from ICN Biomedicals.

    Techniques: Incubation, Radioactivity

    Effect of washing on recovery of BBM proteins. BBMs were treated at 4°C with pronase (300 μg) and washed with cold DPBS (containing protease inhibitors; see Materials and Methods). These washed BBMs were extracted at 4°C with 1% Triton X-100, boiled, and electrophoresed on an SDS–10% polyacrylamide gel. The gel was then stained with Coomassie brilliant blue R-250. First lane, third molecular mass (Mr) markers; second lane, washed BBMs not treated with pronase (control); third lane, washed BBMs treated for 5 min with pronase; fourth lane, washed BBMs treated for 1 h with pronase. The gel shown in this figure is representative of gels used in seven repetitions of this experiment.

    Journal: Infection and Immunity

    Article Title: Characterization of Membrane-Associated Clostridium perfringens Enterotoxin following Pronase Treatment

    doi:

    Figure Lengend Snippet: Effect of washing on recovery of BBM proteins. BBMs were treated at 4°C with pronase (300 μg) and washed with cold DPBS (containing protease inhibitors; see Materials and Methods). These washed BBMs were extracted at 4°C with 1% Triton X-100, boiled, and electrophoresed on an SDS–10% polyacrylamide gel. The gel was then stained with Coomassie brilliant blue R-250. First lane, third molecular mass (Mr) markers; second lane, washed BBMs not treated with pronase (control); third lane, washed BBMs treated for 5 min with pronase; fourth lane, washed BBMs treated for 1 h with pronase. The gel shown in this figure is representative of gels used in seven repetitions of this experiment.

    Article Snippet: Pronase was purchased from Boehringer Mannheim; antipain, chymostatin, leupeptin, pepstatin, phenylmethylsulfonyl fluoride, dimethyl sulfoxide, EDTA, and Azocoll (Azo dye-impregnated collagen) were purchased from Sigma; and AEBSF [4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride] was obtained from ICN Biomedicals.

    Techniques: Staining

    Native PAGE analysis of pronase-treated 125 I-CPE bound to BBMs at 4°C. 125 ). Washes from the BBMs were also collected, lyophilized, and electrophoresed on the same gel (Wash lanes, i.e., lanes 6, 7, 10, 11, 14, and 15). Lanes 1 to 3 show free 125 I-CPE that was or was not treated with pronase and electrophoresed on the same native gel. Lane 1, 125 I-CPE without pronase treatment; lane 2, 125 I-CPE treated for 5 min with pronase; lane 3, 125 I-CPE treated for 1 h with pronase. Migration of small complex and free 125 I-CPE are noted at the left. The gel shown is representative of gels used in six independent repetitions of the experiment. Note that some nonspecific binding samples show significant levels of dye front radioactivity; this result is an artifact due to diffusion of low- M r radioactive material from gel lanes containing pronase-treated specifically bound radioactivity during the native PAGE used in this experiment (data not shown).

    Journal: Infection and Immunity

    Article Title: Characterization of Membrane-Associated Clostridium perfringens Enterotoxin following Pronase Treatment

    doi:

    Figure Lengend Snippet: Native PAGE analysis of pronase-treated 125 I-CPE bound to BBMs at 4°C. 125 ). Washes from the BBMs were also collected, lyophilized, and electrophoresed on the same gel (Wash lanes, i.e., lanes 6, 7, 10, 11, 14, and 15). Lanes 1 to 3 show free 125 I-CPE that was or was not treated with pronase and electrophoresed on the same native gel. Lane 1, 125 I-CPE without pronase treatment; lane 2, 125 I-CPE treated for 5 min with pronase; lane 3, 125 I-CPE treated for 1 h with pronase. Migration of small complex and free 125 I-CPE are noted at the left. The gel shown is representative of gels used in six independent repetitions of the experiment. Note that some nonspecific binding samples show significant levels of dye front radioactivity; this result is an artifact due to diffusion of low- M r radioactive material from gel lanes containing pronase-treated specifically bound radioactivity during the native PAGE used in this experiment (data not shown).

    Article Snippet: Pronase was purchased from Boehringer Mannheim; antipain, chymostatin, leupeptin, pepstatin, phenylmethylsulfonyl fluoride, dimethyl sulfoxide, EDTA, and Azocoll (Azo dye-impregnated collagen) were purchased from Sigma; and AEBSF [4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride] was obtained from ICN Biomedicals.

    Techniques: Clear Native PAGE, Migration, Binding Assay, Radioactivity, Diffusion-based Assay

    Native PAGE analysis of pronase-treated Triton X-100 extracts from BBMs containing 125 I-CPE bound at 4°C. 125 I-CPE was bound to BBMs at 4°C in the absence (− lanes) or the presence (+ lanes) of a 50-fold excess of unlabeled CPE. After the unbound 125 I-CPE was removed, BBMs were extracted with cold DPBS containing 1% Triton X-100 and then treated at 4°C with pronase (300 μg) for 5 min (third and fourth lanes) or 1 h (fifth and sixth lanes); the first and second lanes contain samples that were not pronase treated. After the conclusion of pronase treatment, protease inhibitors were added (see Materials and Methods) and all samples were electrophoresed on a native 6% polyacrylamide gel. The migration of small complex is noted at the left. The gel shown is representative of gels used in six independent repetitions of the experiment.

    Journal: Infection and Immunity

    Article Title: Characterization of Membrane-Associated Clostridium perfringens Enterotoxin following Pronase Treatment

    doi:

    Figure Lengend Snippet: Native PAGE analysis of pronase-treated Triton X-100 extracts from BBMs containing 125 I-CPE bound at 4°C. 125 I-CPE was bound to BBMs at 4°C in the absence (− lanes) or the presence (+ lanes) of a 50-fold excess of unlabeled CPE. After the unbound 125 I-CPE was removed, BBMs were extracted with cold DPBS containing 1% Triton X-100 and then treated at 4°C with pronase (300 μg) for 5 min (third and fourth lanes) or 1 h (fifth and sixth lanes); the first and second lanes contain samples that were not pronase treated. After the conclusion of pronase treatment, protease inhibitors were added (see Materials and Methods) and all samples were electrophoresed on a native 6% polyacrylamide gel. The migration of small complex is noted at the left. The gel shown is representative of gels used in six independent repetitions of the experiment.

    Article Snippet: Pronase was purchased from Boehringer Mannheim; antipain, chymostatin, leupeptin, pepstatin, phenylmethylsulfonyl fluoride, dimethyl sulfoxide, EDTA, and Azocoll (Azo dye-impregnated collagen) were purchased from Sigma; and AEBSF [4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride] was obtained from ICN Biomedicals.

    Techniques: Clear Native PAGE, Migration

    SDS-PAGE analysis of pronase-treated 125 I-CPE bound to BBMs at RT. 125 I-CPE was bound to BBMs at RT in the absence (− lanes, i.e., odd-numbered lanes) or the presence (+ lanes, i.e., even-numbered lanes) of a 50-fold excess of unlabeled CPE. BBMs containing bound 125 I-CPE were then treated at 4°C with pronase (300 μg) for 5 min (membrane [Membr.] lanes 5 and 6) or 1 h (Membr. lanes 9 and 10) or were extracted with 1% Triton X-100 before being treated at 4°C with 300 μg of pronase for 5 min (lanes 13 and 14) or 1 h (lanes 15 and 16). Membr. lanes 1 and 2 represent similarly prepared samples that were not treated with pronase. Samples shown in lanes 1, 2, 5, 6, 9, and 10 were washed with cold DPBS containing protease inhibitors (see Materials and Methods) before extraction. Washes (Wash lanes) were collected from all samples and then lyophilized. Lanes 7 and 8 and 11 and 12 contain wash samples collected after 5 min or 1 h of pronase treatment of BBMs, respectively. All samples were then electrophoresed on an SDS–6% polyacrylamide gel without sample boiling or β-mercaptoethanol treatment. The migration of intact large complex is noted at the left. The gel shown is representative of gels used in six independent repetitions of the experiment.

    Journal: Infection and Immunity

    Article Title: Characterization of Membrane-Associated Clostridium perfringens Enterotoxin following Pronase Treatment

    doi:

    Figure Lengend Snippet: SDS-PAGE analysis of pronase-treated 125 I-CPE bound to BBMs at RT. 125 I-CPE was bound to BBMs at RT in the absence (− lanes, i.e., odd-numbered lanes) or the presence (+ lanes, i.e., even-numbered lanes) of a 50-fold excess of unlabeled CPE. BBMs containing bound 125 I-CPE were then treated at 4°C with pronase (300 μg) for 5 min (membrane [Membr.] lanes 5 and 6) or 1 h (Membr. lanes 9 and 10) or were extracted with 1% Triton X-100 before being treated at 4°C with 300 μg of pronase for 5 min (lanes 13 and 14) or 1 h (lanes 15 and 16). Membr. lanes 1 and 2 represent similarly prepared samples that were not treated with pronase. Samples shown in lanes 1, 2, 5, 6, 9, and 10 were washed with cold DPBS containing protease inhibitors (see Materials and Methods) before extraction. Washes (Wash lanes) were collected from all samples and then lyophilized. Lanes 7 and 8 and 11 and 12 contain wash samples collected after 5 min or 1 h of pronase treatment of BBMs, respectively. All samples were then electrophoresed on an SDS–6% polyacrylamide gel without sample boiling or β-mercaptoethanol treatment. The migration of intact large complex is noted at the left. The gel shown is representative of gels used in six independent repetitions of the experiment.

    Article Snippet: Pronase was purchased from Boehringer Mannheim; antipain, chymostatin, leupeptin, pepstatin, phenylmethylsulfonyl fluoride, dimethyl sulfoxide, EDTA, and Azocoll (Azo dye-impregnated collagen) were purchased from Sigma; and AEBSF [4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride] was obtained from ICN Biomedicals.

    Techniques: SDS Page, Migration

    SDS-PAGE analysis of free 125 I-CPE treated with pronase. 125 I-CPE (0.15 μg), in the presence of 100 μg of bovine serum albumin, was treated at 4°C with pronase (300 μg) for either 5 min (second lane) or 1 h (third lane) before receiving cold DPBS containing protease inhibitors (see Materials and Methods). These samples were then boiled and electrophoresed on an SDS–10% polyacrylamide gel. For comparison, the first lane shows 125 I-CPE that had not been pronase treated. Migrations of molecular mass markers are shown at the right. The gel shown is representative of gels used in 10 independent repetitions of the experiment.

    Journal: Infection and Immunity

    Article Title: Characterization of Membrane-Associated Clostridium perfringens Enterotoxin following Pronase Treatment

    doi:

    Figure Lengend Snippet: SDS-PAGE analysis of free 125 I-CPE treated with pronase. 125 I-CPE (0.15 μg), in the presence of 100 μg of bovine serum albumin, was treated at 4°C with pronase (300 μg) for either 5 min (second lane) or 1 h (third lane) before receiving cold DPBS containing protease inhibitors (see Materials and Methods). These samples were then boiled and electrophoresed on an SDS–10% polyacrylamide gel. For comparison, the first lane shows 125 I-CPE that had not been pronase treated. Migrations of molecular mass markers are shown at the right. The gel shown is representative of gels used in 10 independent repetitions of the experiment.

    Article Snippet: Pronase was purchased from Boehringer Mannheim; antipain, chymostatin, leupeptin, pepstatin, phenylmethylsulfonyl fluoride, dimethyl sulfoxide, EDTA, and Azocoll (Azo dye-impregnated collagen) were purchased from Sigma; and AEBSF [4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride] was obtained from ICN Biomedicals.

    Techniques: SDS Page

    Figure 3. Lung section from a 2 cm-thick specimen stored in 10% neutral buffered formalin for 22 mo. Immunoperoxidase SAB-staining, obtained with anti-BHV-1 serum, pronase-pretreated for 30 min. Bar = 100 μm.

    Journal: Canadian Journal of Veterinary Research

    Article Title: Demonstration of bovine herpesvirus type 1 and Mannheimia haemolytica antigens in specimens stored for up to 22 months in buffered formalin

    doi:

    Figure Lengend Snippet: Figure 3. Lung section from a 2 cm-thick specimen stored in 10% neutral buffered formalin for 22 mo. Immunoperoxidase SAB-staining, obtained with anti-BHV-1 serum, pronase-pretreated for 30 min. Bar = 100 μm.

    Article Snippet: After deparaffinization, endogenous peroxidase activity was blocked by treatment with 0.3% hydrogen peroxide in absolute methanol for 30 min. Sets of tissue sections were then treated with 0.1% pronase (Kaken Chemicals, Tokyo, Japan) in Tris-HCl buffer (0.05 M, pH 7.6) for 5, 10, 20, or 30 min at room temperature.

    Techniques: Staining

    Figure 1. Lung section from a 0.2-cm thick specimen stored in 10% neutral buffered formalin for 22 mo. Immunoperoxidase SAB-staining, obtained with anti-BHV-1 serum, no pronase pretreatment. Bar = 100 μm.

    Journal: Canadian Journal of Veterinary Research

    Article Title: Demonstration of bovine herpesvirus type 1 and Mannheimia haemolytica antigens in specimens stored for up to 22 months in buffered formalin

    doi:

    Figure Lengend Snippet: Figure 1. Lung section from a 0.2-cm thick specimen stored in 10% neutral buffered formalin for 22 mo. Immunoperoxidase SAB-staining, obtained with anti-BHV-1 serum, no pronase pretreatment. Bar = 100 μm.

    Article Snippet: After deparaffinization, endogenous peroxidase activity was blocked by treatment with 0.3% hydrogen peroxide in absolute methanol for 30 min. Sets of tissue sections were then treated with 0.1% pronase (Kaken Chemicals, Tokyo, Japan) in Tris-HCl buffer (0.05 M, pH 7.6) for 5, 10, 20, or 30 min at room temperature.

    Techniques: Staining

    Figure 5. Lung section from a 0.2-cm thick specimen stored in 10% neutral buffered formalin for 22 mo. Immunoperoxidase SAB-staining, obtained with anti- M. haemolytica serum, pronase-pretreated for 30 min. Bar = 100 μm.

    Journal: Canadian Journal of Veterinary Research

    Article Title: Demonstration of bovine herpesvirus type 1 and Mannheimia haemolytica antigens in specimens stored for up to 22 months in buffered formalin

    doi:

    Figure Lengend Snippet: Figure 5. Lung section from a 0.2-cm thick specimen stored in 10% neutral buffered formalin for 22 mo. Immunoperoxidase SAB-staining, obtained with anti- M. haemolytica serum, pronase-pretreated for 30 min. Bar = 100 μm.

    Article Snippet: After deparaffinization, endogenous peroxidase activity was blocked by treatment with 0.3% hydrogen peroxide in absolute methanol for 30 min. Sets of tissue sections were then treated with 0.1% pronase (Kaken Chemicals, Tokyo, Japan) in Tris-HCl buffer (0.05 M, pH 7.6) for 5, 10, 20, or 30 min at room temperature.

    Techniques: Staining

    Figure 4. Lung section from a 0.2-cm thick specimen stored in 10% neutral buffered formalin for 22 mo. Immunoperoxidase SAB-staining, obtained with anti- M. haemolytica serum, no pronase pretreatment. Bar = 100 μm.

    Journal: Canadian Journal of Veterinary Research

    Article Title: Demonstration of bovine herpesvirus type 1 and Mannheimia haemolytica antigens in specimens stored for up to 22 months in buffered formalin

    doi:

    Figure Lengend Snippet: Figure 4. Lung section from a 0.2-cm thick specimen stored in 10% neutral buffered formalin for 22 mo. Immunoperoxidase SAB-staining, obtained with anti- M. haemolytica serum, no pronase pretreatment. Bar = 100 μm.

    Article Snippet: After deparaffinization, endogenous peroxidase activity was blocked by treatment with 0.3% hydrogen peroxide in absolute methanol for 30 min. Sets of tissue sections were then treated with 0.1% pronase (Kaken Chemicals, Tokyo, Japan) in Tris-HCl buffer (0.05 M, pH 7.6) for 5, 10, 20, or 30 min at room temperature.

    Techniques: Staining

    Figure 2. Lung section from a 0.2-cm thick specimen stored in 10% neutral buffered formalin for 22 mo. Immunoperoxidase SAB-staining, obtained with anti-BHV-1 serum, pronase-pretreated for 30 min. Bar = 100 μm.

    Journal: Canadian Journal of Veterinary Research

    Article Title: Demonstration of bovine herpesvirus type 1 and Mannheimia haemolytica antigens in specimens stored for up to 22 months in buffered formalin

    doi:

    Figure Lengend Snippet: Figure 2. Lung section from a 0.2-cm thick specimen stored in 10% neutral buffered formalin for 22 mo. Immunoperoxidase SAB-staining, obtained with anti-BHV-1 serum, pronase-pretreated for 30 min. Bar = 100 μm.

    Article Snippet: After deparaffinization, endogenous peroxidase activity was blocked by treatment with 0.3% hydrogen peroxide in absolute methanol for 30 min. Sets of tissue sections were then treated with 0.1% pronase (Kaken Chemicals, Tokyo, Japan) in Tris-HCl buffer (0.05 M, pH 7.6) for 5, 10, 20, or 30 min at room temperature.

    Techniques: Staining

    Inhibitory activities on B. thailandensis E264 motility on swarm agar of cell-free supernatant of B. pseudomallei 1710a after filtration, protein digestion, or incubation under different conditions. (A) Inhibition zone in retentate and filtrate fractions following filtration through membranes with different molecular weight cut-off sizes. (B) Inhibitory activity of cell-free supernatant of B. pseudomallei 1710a after treatments with proteinase K (left panel) and pronase (right panel), (a) no treatment control, (b) treatment with proteinase K or pronase, (c) Enzymes alone (no supernatants) were spotted onto the agar in the same concentration as a control. (C) Inhibition activity of cell-free supernatant of B. pseudomallei 1710a following incubation at different pH at 37°C for 24 h. (D) Inhibition activity of cell-free supernatant of B. pseudomallei 1710a following incubation at different temperatures for 24 h. (E) Inhibition activity of cell-free supernatant of B. pseudomallei 1710a following incubation at different salt concentrations at 37°C for 24 h. The controls for (A) , (C) , (D) and (E) are B. thailandensis E264 swarming without the inoculation of cell-free supernatant. Plates were incubated at 37°C for 24 h.

    Journal: BMC Microbiology

    Article Title: Competition between Burkholderia pseudomallei and B. thailandensis

    doi: 10.1186/s12866-015-0395-7

    Figure Lengend Snippet: Inhibitory activities on B. thailandensis E264 motility on swarm agar of cell-free supernatant of B. pseudomallei 1710a after filtration, protein digestion, or incubation under different conditions. (A) Inhibition zone in retentate and filtrate fractions following filtration through membranes with different molecular weight cut-off sizes. (B) Inhibitory activity of cell-free supernatant of B. pseudomallei 1710a after treatments with proteinase K (left panel) and pronase (right panel), (a) no treatment control, (b) treatment with proteinase K or pronase, (c) Enzymes alone (no supernatants) were spotted onto the agar in the same concentration as a control. (C) Inhibition activity of cell-free supernatant of B. pseudomallei 1710a following incubation at different pH at 37°C for 24 h. (D) Inhibition activity of cell-free supernatant of B. pseudomallei 1710a following incubation at different temperatures for 24 h. (E) Inhibition activity of cell-free supernatant of B. pseudomallei 1710a following incubation at different salt concentrations at 37°C for 24 h. The controls for (A) , (C) , (D) and (E) are B. thailandensis E264 swarming without the inoculation of cell-free supernatant. Plates were incubated at 37°C for 24 h.

    Article Snippet: The sample was diluted to 1 mg/ml and digested at an enzyme:substrate ratio of 1:10 (w/w) with proteinase K (Invitrogen, Carlsbad, CA, USA) or pronase (Merck, Nottingham, UK) in the relevant buffer as recommended by the manufacturer (proteinase K in PBS at 37°C for 72 h and pronase in PBS containing 10 mM CaCl2 at 37°C for 72 h).

    Techniques: Filtration, Incubation, Inhibition, Molecular Weight, Activity Assay, Concentration Assay

    Identification of Q24DA secondary targets by DARTS assay. Cell lysates from C7258 were incubated with Q24DA, an inactive structural analog (SR27911), or DMSO (vehicle control); digested with pronase; and subjected to two-dimensional gel electrophoresis

    Journal: Antimicrobial Agents and Chemotherapy

    Article Title: A Quinazoline-2,4-Diamino Analog Suppresses Vibrio cholerae Flagellar Motility by Interacting with Motor Protein PomB and Induces Envelope Stress

    doi: 10.1128/AAC.00473-13

    Figure Lengend Snippet: Identification of Q24DA secondary targets by DARTS assay. Cell lysates from C7258 were incubated with Q24DA, an inactive structural analog (SR27911), or DMSO (vehicle control); digested with pronase; and subjected to two-dimensional gel electrophoresis

    Article Snippet: For DARTS assays, 800 μg of protein was reacted with 16 μg of Q24DA dissolved in DMSO for 30 min at 37°C, and the mixture was subsequently treated with pronase (1 mg/ml; Promega) for 30 min at 42°C.

    Techniques: Incubation, Two-Dimensional Gel Electrophoresis, Electrophoresis