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Proteolytic activity profile of secreted acid proteases from P. brasiliensis . ( A )Yeast cells were grown in YPDm with a pH of 6.5 or 4 for five days. Proteolytic activity was measured using 20 µL of sample (cell-free supernatant) and 80 µL of 7.5 µM bovine serum albumin (BSA) in 0.1 M citric acid/sodium phosphate, with a pH of 3.3, and incubated at 50 °C. Samples were incubated in the presence or absence of Pepstatin A (5, 10, and 15 μM) or PMSF (100 μM). Then, 20 µL were removed after 30 min and added to 180 µL of <t>Coomassie</t> <t>PlusTM</t> protein assay reagent (Pierce) in a microtiter plate. Absorbance was read at 590 nm, and activity (IU) was calculated relative to BSA degradation using a standard curve. One IU is defined as the degradation of 1 µmol of BSA min −1 .mL −1 . ( B ) Samples were incubated with collagen or hemoglobin for 30 min in the presence or absence of Pepstatin A. ** p ≤ 0.01, and *** p ≤ 0.001. The results shown are representative of three independent experiments.
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Proteolytic activity profile of secreted acid proteases from P. brasiliensis . ( A )Yeast cells were grown in YPDm with a pH of 6.5 or 4 for five days. Proteolytic activity was measured using 20 µL of sample (cell-free supernatant) and 80 µL of 7.5 µM bovine serum albumin (BSA) in 0.1 M citric acid/sodium phosphate, with a pH of 3.3, and incubated at 50 °C. Samples were incubated in the presence or absence of Pepstatin A (5, 10, and 15 μM) or PMSF (100 μM). Then, 20 µL were removed after 30 min and added to 180 µL of <t>Coomassie</t> <t>PlusTM</t> protein assay reagent (Pierce) in a microtiter plate. Absorbance was read at 590 nm, and activity (IU) was calculated relative to BSA degradation using a standard curve. One IU is defined as the degradation of 1 µmol of BSA min −1 .mL −1 . ( B ) Samples were incubated with collagen or hemoglobin for 30 min in the presence or absence of Pepstatin A. ** p ≤ 0.01, and *** p ≤ 0.001. The results shown are representative of three independent experiments.
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Proteolytic activity profile of secreted acid proteases from P. brasiliensis . ( A )Yeast cells were grown in YPDm with a pH of 6.5 or 4 for five days. Proteolytic activity was measured using 20 µL of sample (cell-free supernatant) and 80 µL of 7.5 µM bovine serum albumin (BSA) in 0.1 M citric acid/sodium phosphate, with a pH of 3.3, and incubated at 50 °C. Samples were incubated in the presence or absence of Pepstatin A (5, 10, and 15 μM) or PMSF (100 μM). Then, 20 µL were removed after 30 min and added to 180 µL of <t>Coomassie</t> <t>PlusTM</t> protein assay reagent (Pierce) in a microtiter plate. Absorbance was read at 590 nm, and activity (IU) was calculated relative to BSA degradation using a standard curve. One IU is defined as the degradation of 1 µmol of BSA min −1 .mL −1 . ( B ) Samples were incubated with collagen or hemoglobin for 30 min in the presence or absence of Pepstatin A. ** p ≤ 0.01, and *** p ≤ 0.001. The results shown are representative of three independent experiments.
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Proteolytic activity profile of secreted acid proteases from P. brasiliensis . ( A )Yeast cells were grown in YPDm with a pH of 6.5 or 4 for five days. Proteolytic activity was measured using 20 µL of sample (cell-free supernatant) and 80 µL of 7.5 µM bovine serum albumin (BSA) in 0.1 M citric acid/sodium phosphate, with a pH of 3.3, and incubated at 50 °C. Samples were incubated in the presence or absence of Pepstatin A (5, 10, and 15 μM) or PMSF (100 μM). Then, 20 µL were removed after 30 min and added to 180 µL of <t>Coomassie</t> <t>PlusTM</t> protein assay reagent (Pierce) in a microtiter plate. Absorbance was read at 590 nm, and activity (IU) was calculated relative to BSA degradation using a standard curve. One IU is defined as the degradation of 1 µmol of BSA min −1 .mL −1 . ( B ) Samples were incubated with collagen or hemoglobin for 30 min in the presence or absence of Pepstatin A. ** p ≤ 0.01, and *** p ≤ 0.001. The results shown are representative of three independent experiments.
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Proteolytic activity profile of secreted acid proteases from P. brasiliensis . ( A )Yeast cells were grown in YPDm with a pH of 6.5 or 4 for five days. Proteolytic activity was measured using 20 µL of sample (cell-free supernatant) and 80 µL of 7.5 µM bovine serum albumin (BSA) in 0.1 M citric acid/sodium phosphate, with a pH of 3.3, and incubated at 50 °C. Samples were incubated in the presence or absence of Pepstatin A (5, 10, and 15 μM) or PMSF (100 μM). Then, 20 µL were removed after 30 min and added to 180 µL of <t>Coomassie</t> <t>PlusTM</t> protein assay reagent (Pierce) in a microtiter plate. Absorbance was read at 590 nm, and activity (IU) was calculated relative to BSA degradation using a standard curve. One IU is defined as the degradation of 1 µmol of BSA min −1 .mL −1 . ( B ) Samples were incubated with collagen or hemoglobin for 30 min in the presence or absence of Pepstatin A. ** p ≤ 0.01, and *** p ≤ 0.001. The results shown are representative of three independent experiments.
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Proteolytic activity profile of secreted acid proteases from P. brasiliensis . ( A )Yeast cells were grown in YPDm with a pH of 6.5 or 4 for five days. Proteolytic activity was measured using 20 µL of sample (cell-free supernatant) and 80 µL of 7.5 µM bovine serum albumin (BSA) in 0.1 M citric acid/sodium phosphate, with a pH of 3.3, and incubated at 50 °C. Samples were incubated in the presence or absence of Pepstatin A (5, 10, and 15 μM) or PMSF (100 μM). Then, 20 µL were removed after 30 min and added to 180 µL of <t>Coomassie</t> <t>PlusTM</t> protein assay reagent (Pierce) in a microtiter plate. Absorbance was read at 590 nm, and activity (IU) was calculated relative to BSA degradation using a standard curve. One IU is defined as the degradation of 1 µmol of BSA min −1 .mL −1 . ( B ) Samples were incubated with collagen or hemoglobin for 30 min in the presence or absence of Pepstatin A. ** p ≤ 0.01, and *** p ≤ 0.001. The results shown are representative of three independent experiments.
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Proteolytic activity profile of secreted acid proteases from P. brasiliensis . ( A )Yeast cells were grown in YPDm with a pH of 6.5 or 4 for five days. Proteolytic activity was measured using 20 µL of sample (cell-free supernatant) and 80 µL of 7.5 µM bovine serum albumin (BSA) in 0.1 M citric acid/sodium phosphate, with a pH of 3.3, and incubated at 50 °C. Samples were incubated in the presence or absence of Pepstatin A (5, 10, and 15 μM) or PMSF (100 μM). Then, 20 µL were removed after 30 min and added to 180 µL of <t>Coomassie</t> <t>PlusTM</t> protein assay reagent (Pierce) in a microtiter plate. Absorbance was read at 590 nm, and activity (IU) was calculated relative to BSA degradation using a standard curve. One IU is defined as the degradation of 1 µmol of BSA min −1 .mL −1 . ( B ) Samples were incubated with collagen or hemoglobin for 30 min in the presence or absence of Pepstatin A. ** p ≤ 0.01, and *** p ≤ 0.001. The results shown are representative of three independent experiments.
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Proteolytic activity profile of secreted acid proteases from P. brasiliensis . ( A )Yeast cells were grown in YPDm with a pH of 6.5 or 4 for five days. Proteolytic activity was measured using 20 µL of sample (cell-free supernatant) and 80 µL of 7.5 µM bovine serum albumin (BSA) in 0.1 M citric acid/sodium phosphate, with a pH of 3.3, and incubated at 50 °C. Samples were incubated in the presence or absence of Pepstatin A (5, 10, and 15 μM) or PMSF (100 μM). Then, 20 µL were removed after 30 min and added to 180 µL of <t>Coomassie</t> <t>PlusTM</t> protein assay reagent (Pierce) in a microtiter plate. Absorbance was read at 590 nm, and activity (IU) was calculated relative to BSA degradation using a standard curve. One IU is defined as the degradation of 1 µmol of BSA min −1 .mL −1 . ( B ) Samples were incubated with collagen or hemoglobin for 30 min in the presence or absence of Pepstatin A. ** p ≤ 0.01, and *** p ≤ 0.001. The results shown are representative of three independent experiments.
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Image Search Results


Proteolytic activity profile of secreted acid proteases from P. brasiliensis . ( A )Yeast cells were grown in YPDm with a pH of 6.5 or 4 for five days. Proteolytic activity was measured using 20 µL of sample (cell-free supernatant) and 80 µL of 7.5 µM bovine serum albumin (BSA) in 0.1 M citric acid/sodium phosphate, with a pH of 3.3, and incubated at 50 °C. Samples were incubated in the presence or absence of Pepstatin A (5, 10, and 15 μM) or PMSF (100 μM). Then, 20 µL were removed after 30 min and added to 180 µL of Coomassie PlusTM protein assay reagent (Pierce) in a microtiter plate. Absorbance was read at 590 nm, and activity (IU) was calculated relative to BSA degradation using a standard curve. One IU is defined as the degradation of 1 µmol of BSA min −1 .mL −1 . ( B ) Samples were incubated with collagen or hemoglobin for 30 min in the presence or absence of Pepstatin A. ** p ≤ 0.01, and *** p ≤ 0.001. The results shown are representative of three independent experiments.

Journal: Journal of Fungi

Article Title: Characterization of Aspartic Proteases from Paracoccidioides brasiliensis and Their Role in Fungal Thermo-Dimorphism

doi: 10.3390/jof9030375

Figure Lengend Snippet: Proteolytic activity profile of secreted acid proteases from P. brasiliensis . ( A )Yeast cells were grown in YPDm with a pH of 6.5 or 4 for five days. Proteolytic activity was measured using 20 µL of sample (cell-free supernatant) and 80 µL of 7.5 µM bovine serum albumin (BSA) in 0.1 M citric acid/sodium phosphate, with a pH of 3.3, and incubated at 50 °C. Samples were incubated in the presence or absence of Pepstatin A (5, 10, and 15 μM) or PMSF (100 μM). Then, 20 µL were removed after 30 min and added to 180 µL of Coomassie PlusTM protein assay reagent (Pierce) in a microtiter plate. Absorbance was read at 590 nm, and activity (IU) was calculated relative to BSA degradation using a standard curve. One IU is defined as the degradation of 1 µmol of BSA min −1 .mL −1 . ( B ) Samples were incubated with collagen or hemoglobin for 30 min in the presence or absence of Pepstatin A. ** p ≤ 0.01, and *** p ≤ 0.001. The results shown are representative of three independent experiments.

Article Snippet: Then, 20 μL were removed after 5, 15, 30, or 45 min and added to 180 μL of a Coomassie PlusTM protein assay reagent (Pierce) in a microtiter plate.

Techniques: Activity Assay, Incubation