Yeast extract, malt extract, peptone, glucose, microbiological agar, Hepes, Triton X-100, CaCl2, and 2-mercaptoethanol were obtained from Merck (Johannesburg, GP, South Africa). Yeast nitrogen base (YNB) and PierceTM Colorimetric Protease Assay Kit were obtained from Thermo Fisher Scientific (Johannesburg, GP, South Africa); 50 mL centrifuge tubes and 2 mL plastic tubes were obtained from Lasec (Johannesburg, GP, South Africa), as well as a haemocytometer (Marienfeld, BW, Germany). Black, sterile disposable 96-well flat-bottom microtiter plates were obtained from Greiner Bio-One (Frickenhausen, BW, Germany). Recombinant furin was acquired from New England Biolabs (Ipswich, MA, USA). The mimetic peptide ASYQTQTNSPRRA RSVASQS (corresponding to the amino acid sequence of SARS-CoV-2 S1/S2) containing the 7-methoxycoumarin-4-yl acetyl/2,4-dinitrophenyl (MCA/DNP) FRET pair was synthesised by Biomatik (Wilmington, DE, USA). VICTOR Nivo multimode microplate reader was purchased from PerkinElmer (Waltham, MA, USA).
Pierce colorimetric protease assay kit
Manufactured by Thermo Fisher Scientific
Sourced in Germany, United Kingdom
The Pierce™ Colorimetric Protease Assay Kit is a laboratory product designed to quantify protease activity in samples. It uses a chromogenic substrate that changes color upon cleavage by proteases, allowing for colorimetric detection and measurement of protease levels.
Automatically generated - may contain errors
Lab products found in correlation
Recombinant furin, by New England Biolabs (1 mentions)
Yeast nitrogen base, by Thermo Fisher Scientific (1 mentions)
Peptone, by Merck Group (1 mentions)
Victor nivo multimode microplate reader, by PerkinElmer (1 mentions)
Amicon ultra, by Merck Group (1 mentions)
Peroxidase activity assay kit, by Merck Group (1 mentions)
Dextrose, by Merck Group (1 mentions)
5 protocols using pierce colorimetric protease assay kit
1
SARS-CoV-2 Furin Cleavage Site Assay
2
Protease Inhibition Activity of rhSLPI
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A 1 x 10 5 cells/ml sample containing pCMV-SLPI-HA, mock transfection, were cultured in a 6-well plate with DMEM complete medium supplemented with 500 µg/ml hygromycin B at 37 o C, 5% CO 2 , and 95% O 2 for 2 days. The culture medium was then collected and concentrated 40fold using an Amicon Ultra filter device (Merck, Germany). The protease inhibitory activity of the rhSLPI concentrated protein was determined using a Pierce TM Colorimetric Protease Assay Kit (Thermo Fisher Scientific Inc., United Kingdom). Briefly, 100 µl of succinylated casein solution was added to microplate wells. Consequently, 100 µl of assay buffer was applied to the microplate wells followed by 50 µl of 125 µg/ml trypsin or 50 µl of sample mixture (containing 5 µl, 15 µl, and 30 µl concentrated culture medium and trypsin; the final concentration of trypsin in the mixture was 125 µg/ml). The mixed solution was incubated for 20 min at room temperature, and then TNBSA working solution was applied to each well. Then, the microplate was incubated for 20 min at room temperature. The color of the solution was determined by the spectrophotometric method at λ 450 nm. The activity of the rhSLPI was in inverse proportion to the color intensity.
3
Enzyme Activity Characterization of Fungal Secretome
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The fungal biomass was separated by filtration, and the filtrates were further clarified by centrifugation at 8000 g, 4 °C for 20 min. Secreted proteins in the culture were detected by a modified Bradford’s method68 (link),69 . Standards were prepared using 0.2–2 mg/mL of BSA solution.
Cellulase activities of the supernatants were determined according to the protocols described by Ghose et al.70 (link). Total cellulase (FPase), endoglucanase (CMCase) and xylanase activities were quantified in terms of reducing sugar (glucose/xylose) released using the DNS assay71 (link). BGL activity was quantified in terms of micromoles of pNPG released under standard assay conditions. To determine glucose tolerance of the BGLs, citrate–phosphate buffer (50 mM, pH 4.8) containing varying concentrations of glucose was used in the assay.
Peroxidase activity (an indicator of ligninolytic action) was determined using the Peroxidase Activity Assay Kit (MAK092) from Sigma-Aldrich, as per the manufacturer’s protocol for colorimetric tests. Protease activity was determined using the Pierce™ Colorimetric Protease Assay Kit (Catalog number: 23263) from ThermoFisher Scientific, according to the manufacturer’s protocol.
The results of enzyme assays presented in Figs.1 and 3 are the average of biological triplicates.
Cellulase activities of the supernatants were determined according to the protocols described by Ghose et al.70 (link). Total cellulase (FPase), endoglucanase (CMCase) and xylanase activities were quantified in terms of reducing sugar (glucose/xylose) released using the DNS assay71 (link). BGL activity was quantified in terms of micromoles of pNPG released under standard assay conditions. To determine glucose tolerance of the BGLs, citrate–phosphate buffer (50 mM, pH 4.8) containing varying concentrations of glucose was used in the assay.
Peroxidase activity (an indicator of ligninolytic action) was determined using the Peroxidase Activity Assay Kit (MAK092) from Sigma-Aldrich, as per the manufacturer’s protocol for colorimetric tests. Protease activity was determined using the Pierce™ Colorimetric Protease Assay Kit (Catalog number: 23263) from ThermoFisher Scientific, according to the manufacturer’s protocol.
The results of enzyme assays presented in Figs.
4
Aspergillus fumigatus Protease Mobilization
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A. fumigatus strain was cultured on potato dextrose agar slants (PDA) agar (200 g/L potato infusion, 20 g/L dextrose, 20 g/L agar (Merck, South Africa)) and incubated for 24 h at 30 °C. Following this, a loopful of fungal colonies were picked and inoculated into a 250 mL conical flask that contained 100 mL of fresh, sterile YNB (6.7 g/L; Thermo Fisher Scientific, South Africa) broth that was supplemented with glucose (4%; w/v; Merck, South Africa). Next, the flask was then incubated at 30 °C for 32 h while agitated at 160 rpm on an orbital shaker (Lasec, South Africa). After 32 h, 25 mL of the culture media was then dispensed into a 50 mL centrifuge tube. The tube was centrifuged at 1000 × g for 5 min at 30 °C to pellet the cells and mobilise the Aspergillus protease(s) into the supernatant. To confirm the mobilisation of the protease(s) into the supernatant, it was tested with a Pierce™ Colorimetric Protease Assay Kit (Thermo Fisher Scientific) in accordance with the manufacturer’s protocol. The Kit detects the total protease activity in the tested sample.
5
Comparative Protease Analysis in C. sporogenes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To detect and compare the levels of proteases in wild-type or recombinant C. sporogenes strains, gelatin zymography was performed using 10% Novex® Zymogram Gel according to manufacturer’s instructions (MAN0005885, Novex/Invitrogen). To do this, samples from cultures of C. sporogenes-NT-ΔpyrE and C. sporogenes-NT-ΔpyrEΔPR1 were taken at four different time points (5h, 7h,10h, and 12h), centrifuged (10,000 x g, 10 min) and filter sterilised (0.22 μm syringe filter). Sterile supernatants, normalised according to cell density (OD600 = 1.2), were mixed with Tris-Glycine SDS Sample Buffer (2x), of which 20 μl was loaded under non-reducing conditions into precast gelatin zymography gels. Clostridium butyricum DSM 10702 supernatant sample, obtained from 10-hour growth, was used as a control. The zymograph was run at 125 V for 90 minutes.
To quantify the total protease activity in culture supernatants, a colorimetric protease assay was performed in 96-well plates, according to manufacturer’s recommendations (23263, Thermo Scientific™ Pierce™ Colorimetric Protease Assay Kit). The spectrophotometric measurement was recorded using a microplate reader (iD3, SpectraMax). Comparative protease activities were calculated and expressed in ng/ml using trypsin standard curve as a reference.
To quantify the total protease activity in culture supernatants, a colorimetric protease assay was performed in 96-well plates, according to manufacturer’s recommendations (23263, Thermo Scientific™ Pierce™ Colorimetric Protease Assay Kit). The spectrophotometric measurement was recorded using a microplate reader (iD3, SpectraMax). Comparative protease activities were calculated and expressed in ng/ml using trypsin standard curve as a reference.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!