Osteochondral tissue constructs (N = 3, n = 3) were harvested and dissected, as previously described. The ALP activity and quantity of DNA was determined using a protocol modified from the literature [18 ]. The bone and cartilage ends of the tissue were homogenized separately using a TissueLyser II with ALP lysis buffer, consisting of 1 mM MgCl2 (Sigma Aldrich, UK), 20 μM ZnCl2 (Sigma Aldrich, UK) and 0.1% (w/v) octyl-β-glucopyranoside (Sigma Aldrich, UK) in 10 mM tris(hydroxymethyl)aminomethane buffer (pH 7.4) (Sigma Aldrich, UK) with the sample lysate immediately stored at −80 °C. To perform the assay, the samples were thawed on ice and then each sample was incubated with p-nitrophenol phosphate (Sigma Aldrich, UK) at 37 °C for 30 min. The reaction was terminated using 1 N NaOH and the absorbance measured at 405 nm using a SpectraMax M5 plate reader. A standard curve between 0 and 800 μM of p-nitrophenol phosphate was used to calculate the sample activity. An equivalent volume from the remainder of the sample was then used for DNA quantification using a PicoGreen™ assay (Thermo Fisher, UK), according to the manufacturer's protocol. The fluorescence was measured at 485/535 nm using a SpectraMax M5 plate reader. The ALP activity normalized to DNA quantity was compared between the bone and cartilage regions.
Octyl β glucopyranoside
Manufactured by Merck Group
Sourced in United Kingdom, United States
Octyl-β-glucopyranoside is a non-ionic detergent used in the solubilization and purification of membrane proteins. It is a glucose-based surfactant with an octyl (C8) hydrophobic chain.
Automatically generated - may contain errors
Lab products found in correlation
Picogreen assay, by Thermo Fisher Scientific (2 mentions)
P nitrophenol phosphate, by Merck Group (2 mentions)
Tris hydroxymethyl aminomethane buffer ph 7.4, by Merck Group (2 mentions)
Mgcl2, by Merck Group (2 mentions)
Zncl2, by Merck Group (2 mentions)
Tissuelyser 2, by Merck Group (1 mentions)
Beh c18 packing material, by Waters Corporation (1 mentions)
Dithiothreitol (dtt), by Promega (1 mentions)
Urea, by Teknova (1 mentions)
Trifluoroacetic acid tfa, by Thermo Fisher Scientific (1 mentions)
Z fr amc, by Bachem (1 mentions)
Acetonitrile acn, by Thermo Fisher Scientific (1 mentions)
Biacore 3000, by Cytiva (1 mentions)
Enhanced chemi luminescence (ecl), by GE Healthcare (1 mentions)
Trans blot sd semi dry transfer cell, by Bio-Rad (1 mentions)
α cyano 4 hydroxycinnamic acid matrix, by Merck Group (1 mentions)
Ethanol, by Merck Group (1 mentions)
Chloroform, by Merck Group (1 mentions)
Glacial acetic acid, by Merck Group (1 mentions)
Acetonitrile, by Honeywell (1 mentions)
6 protocols using octyl β glucopyranoside
1
Osteochondral Tissue ALP and DNA Assay
2
Quantifying Osteochondral Tissue ALP Activity
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Osteochondral tissue constructs (N = 3, n = 3) were harvested and dissected, as previously described. The ALP activity and quantity of DNA was determined using a protocol modified from the literature [18 ]. The bone and cartilage ends of the tissue were homogenized separately using a TissueLyser II with ALP lysis buffer, consisting of 1 mM MgCl2 (Sigma Aldrich, UK), 20 μM ZnCl2 (Sigma Aldrich, UK) and 0.1% (w/v) octyl-β-glucopyranoside (Sigma Aldrich, UK) in 10 mM tris(hydroxymethyl)aminomethane buffer (pH 7.4) (Sigma Aldrich, UK) with the sample lysate immediately stored at −80 °C. To perform the assay, the samples were thawed on ice and then each sample was incubated with p-nitrophenol phosphate (Sigma Aldrich, UK) at 37 °C for 30 min. The reaction was terminated using 1 N NaOH and the absorbance measured at 405 nm using a SpectraMax M5 plate reader. A standard curve between 0 and 800 μM of p-nitrophenol phosphate was used to calculate the sample activity. An equivalent volume from the remainder of the sample was then used for DNA quantification using a PicoGreen™ assay (Thermo Fisher, UK), according to the manufacturer's protocol. The fluorescence was measured at 485/535 nm using a SpectraMax M5 plate reader. The ALP activity normalized to DNA quantity was compared between the bone and cartilage regions.
3
Proteases and Peptide Cleavage Assays
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Human recombinant proteases (with catalogue numbers indicated)
were obtained from R&D Systems (Minneapolis, MN), consisting of
human recombinant cathepsin L (#952-CY-010), human recombinant cathepsin
V (#1080-CY-010), human recombinant PC1/3 (#2810-SE-010), and human
recombinant PC2 (#6018-SE-010). Protease MSP-MS assays utilized the
library of 228 14-mer peptides designed to contain all possible protease
cleavage sites, as previously described29 (link),30 (link) (peptides
synthesized by Anaspec, Fremont, CA). Assays utilized octyl-β-glucopyranoside
(Sigma-Aldrich, Darmstadt, Germany), dithiothreitol (DTT, Promega,
Madison, WI), BEH C18 packing material (Waters Corporation, Milford,
MA), acetonitrile (ACN, Fisher Chemical, Pittsburgh, PA), trifluoroacetic
acid (TFA, Fisher Chemical, Pittsburgh, PA), urea (Teknova, Hollister,
CA), and C18 for solid-phase extraction (SPE) stage-tips (3M, Maplewood,
MN). Fluorogenic proteolytic assays used the substrates Z-K-R-AMC,
Z-R-K-AMC, Z-K-K-AMC, Z-R-R-AMC, Z-L-K-R-AMC, Z-W-K-R-AMC, Z-F-K-R-AMC,
Z-Y-K-R-AMC, Z-V-K-R-AMC, Z-G-K-R-AMC, and Z-A-K-R-AMC from Genscript
(Piscataway, NH); Z-F-R-AMC was from Bachem (Vista, CA) and pERTKR-AMC
was from R&D Systems.
were obtained from R&D Systems (Minneapolis, MN), consisting of
human recombinant cathepsin L (#952-CY-010), human recombinant cathepsin
V (#1080-CY-010), human recombinant PC1/3 (#2810-SE-010), and human
recombinant PC2 (#6018-SE-010). Protease MSP-MS assays utilized the
library of 228 14-mer peptides designed to contain all possible protease
cleavage sites, as previously described29 (link),30 (link) (peptides
synthesized by Anaspec, Fremont, CA). Assays utilized octyl-β-glucopyranoside
(Sigma-Aldrich, Darmstadt, Germany), dithiothreitol (DTT, Promega,
Madison, WI), BEH C18 packing material (Waters Corporation, Milford,
MA), acetonitrile (ACN, Fisher Chemical, Pittsburgh, PA), trifluoroacetic
acid (TFA, Fisher Chemical, Pittsburgh, PA), urea (Teknova, Hollister,
CA), and C18 for solid-phase extraction (SPE) stage-tips (3M, Maplewood,
MN). Fluorogenic proteolytic assays used the substrates Z-K-R-AMC,
Z-R-K-AMC, Z-K-K-AMC, Z-R-R-AMC, Z-L-K-R-AMC, Z-W-K-R-AMC, Z-F-K-R-AMC,
Z-Y-K-R-AMC, Z-V-K-R-AMC, Z-G-K-R-AMC, and Z-A-K-R-AMC from Genscript
(Piscataway, NH); Z-F-R-AMC was from Bachem (Vista, CA) and pERTKR-AMC
was from R&D Systems.
4
Pneumolysin Binding Kinetics Assay
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The affinity and kinetics of pneumolysin, its mutants and cholesterol or β-sitosterol (both as liposome) were measured by SPR at 25 °C on a BIAcore® 3000 using CM5 chips. Pneumolysin or its T459G/L460G mutant was dissolved in 10 mM sodium acetate (pH 4.0) and immobilized on the CM5 chip with 1000 response units (RU) at a flow rate of 10 μL/min. Liposomes containing cholesterol or β-sitosterol were serially diluted in PBST buffer (PBS containing 0.005% Tween 20) to concentrations ranging from 20 μM to 1.25 μM. Each of the five concentrations used was injected at a flow rate of 30 μL/min for 2 min; for dissociation, the flow rate was set at 30 μL/min for 6 min. To regenerate channels, 40 mM β-Octyl glucopyranoside (Sigma-Aldrich St. Louis, MO, USA) was injected for 90 sec at a flow rate of 20 μL/min, followed by injection of PBST buffer for 90 s until the RU reached the original reading. All injections were performed at 25 °C. The data was fitted with a 1:1 binding model using BIA evaluation 4.1. Figures were made using Prism (GraphPad Software, Inc.).
5
Immunodetection of PfPAP2 Membrane Protein
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For immunodetection of PfPAP2, we generated antibody in mice against rAPD domain. We labeled this antibody as anti-PfPAP2 antibody. PfPAP2 is an integral membrane protein so we isolated the membrane fraction of parasite using RIPA buffer and then solubilized it with 1% w/v of β-octylglucopyranoside (Sigma, USA). Proteins were separated by SDS-PAGE, transferred to nitrocellulose membrane using Trans-Blot SD Semi-Dry Transfer Cell (Bio-Rad, USA). The membrane was probed with anti-PfPAP2 mouse sera at a dilution of 1:200 and developed using ECL (enhanced chemiluminescence) Plus Western Blotting Detection system kit (GE Healthcare, USA) following the manufacturer’s specifications. Pre-immune sera were used as negative control.
6
Microscopic Slide Preparation and Reagents
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Superfrost TM microscopic slides were purchased from Thermo Fisher (USA).
Analytical grade ethanol, glacial acetic acid, chloroform and red phosphorous were obtained from Merck (Germany). Sequencing grade bovine trypsin, β-octyl glucopyranoside and α-Cyano-4-hydroxycinnamic acid matrix were purchased from Sigma Aldrich (St. Louis, MO).
Water and acetonitrile were of LC-MS grade and obtained from Honeywell (California, USA). All other chemicals used were of analytical grade.
Analytical grade ethanol, glacial acetic acid, chloroform and red phosphorous were obtained from Merck (Germany). Sequencing grade bovine trypsin, β-octyl glucopyranoside and α-Cyano-4-hydroxycinnamic acid matrix were purchased from Sigma Aldrich (St. Louis, MO).
Water and acetonitrile were of LC-MS grade and obtained from Honeywell (California, USA). All other chemicals used were of analytical grade.
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