The optimisation of synthetic conditions was conducted by monitoring the absorbance at 400 nm. Typically, the activity assay was conducted in the optimised pH 9.0 with a fixed concentration of 0.8 mM BNPP and 0.5 mg/mL catalyst prepared at different conditions with at least 3 parallel samples. After incubation for 3 h at 37 °C, the absorbance at 400 nm was recorded by Tecan Pro 200 Microplate Reader for mass activity comparison. The specific activity comparison was normalised by BET surface area in Supplementary Table 6 . Due to the presence of high-energy phosphate bond in BNPP, all the data have been treated to subtract the autolysis of BNPP and the background of catalysts.
Pro 200 microplate reader
Manufactured by Tecan
The Pro 200 Microplate Reader is a versatile laboratory instrument designed for quantitative analysis of samples in microplates. It measures the absorbance of samples at user-selectable wavelengths, enabling various colorimetric assays to be performed.
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Lab products found in correlation
7 protocols using pro 200 microplate reader
1
Optimisation of BNPP Hydrolysis Catalysts
2
Enzymatic Cleavage Kinetics Assay
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4-nitrophenyl N-acetyl-β-d -glucosaminide was chosen to study the cleavage effect in different buffers. Typically, 200 µL of solutions containing 5 mM 4-nitrophenyl N-acetyl-β-d -glucosaminide and 1 mg/mL Ce-FMA-FA-RT-20 were incubated in different buffers at 60 °C for 8 h. Three parallel samples were conducted each time. After 8 h, 180 µL of the solution was added into 96-well plate and measured by Tecan Pro 200 Microplate Reader at 405 nm.
3
Crystal Violet Biofilm Dispersion Assay
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The biofilm dispersion was similar as above without covering Nest circle microscope cover glass in 24-well plate. After fixation, wells were stained with 150 µL of 0.1%wt crystal violet for 10 min and then washed with 200 µL of water to remove floating colour. After drying, 250 µL of ethanol was used to elute the crystal violet of each well. The stained solution was diluted moderately in 96-well plate for further measure at the absorbance at 590 nm by Tecan Pro 200 Microplate Reader.
4
Enzymatic Cleavage Kinetics Study
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2-nitrophenyl β-d -galactopyranoside was chosen to study the cleavage effect in different buffers. Typically, 200 µL of solutions containing 5 mM 2-nitrophenyl β-d -galactopyranoside and 1 mg/mL Ce-FMA-FA-RT-20 were incubated in different buffers at 60 °C for 8 h. Three parallel samples were conducted each time. After 8 h, 180 µL of the solution was added into 96-well plate and measured by Tecan Pro 200 Microplate Reader at 420 nm.
5
Phosphatase activity assay optimization
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Typically, the activity assays were conducted in the optimised pH 10.0 with a fixed concentration of 10 mM pNPP and 0.5 mg/mL catalyst prepared at different conditions with 8 parallel samples. After incubation for 20 min at 37 °C, the absorbance at 400 nm was recorded by Tecan Pro 200 Microplate Reader for mass activity comparison. The specific activity comparison was normalised by BET surface area in Supplementary Table 6 . Due to the presence of high-energy phosphate bond in pNPP, all the data have been treated to subtract the autolysis of pNPP and the background of catalysts. The maximum and minimum were removed when plotting.
6
Triglyceride Quantification in Fish Muscle
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Skeletal muscle was assayed for triglyceride content following the commercial triglyceride assay kit (#65336—Sigma) on a 200 PRO microplate reader (TECAN). In brief, overnight fasted surface fish and Pachón cavefish were euthanized via ms222 (IACUC, 2021-129), and skeletal muscle immediately posterior to the dorsal fin was dissected. Then, 100 mg of skeletal muscle was washed with cold 1× PBS and homogenized in 1 mL of 5% NP-40/ddH2O via 20 strokes with a pestle. Samples were gradually heated from 80 °C to 100 °C within a 5-min timespan and then cooled at room temperature. This process was repeated two times followed by centrifugation at 16,900 × g for 2 min. Samples were diluted 10-fold in ddH2O. The samples were incubated in provided lipase for 20 min at room temperature followed by the addition of the triglyceride reaction mix and incubated at room temperature for 60 min. Samples were then measured at OD 570 nm. Due to extremely high triglyceride levels in all Pachón samples, repeated dilutions were needed to remain within the working range. However, three Pachón cavefish remained out of range (high triglyceride content) and were thus removed from triglyceride quantification. This did not interfere with the final interpretation of triglyceride levels between surface fish and Pachón cavefish.
7
Measurement of Phosphoglucomutase Activity
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Skeletal muscle from overnight fasted fish was extracted posterior to the dorsal fin and immediately frozen in liquid nitrogen. Frozen skeletal muscle was homogenized in the provided buffer with the Phosphoglucomutase activity assay kit (ab155896 abcam) supplemented with protease and phosphatase inhibitors (CIP-treated samples contained only protease inhibitors). Samples were then gently agitated at 4 °C for 30 min, spun down, and protein measured using the Pierce BCA Protein Assay Kit (Thermo #23227). For CIP incubation, 10 µg of protein from each sample was incubated in 100 µL 1× CIP buffer and 2 µL CIP (5,000 U/mL) (M0525S NEG) for 30 min at 37 °C. Phosphoglucomutase activity of CIP-treated and nontreated samples were then measured according to the manufacturer’s instructions (ab155896 abcam) using a 200 PRO microplate reader (TECAN).
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