Ezq protein quantification kit

Coral fragments that were incubated for 48 h were subjected to proteomic analysis. Previously frozen samples were thawed and tissue was removed from the calcium carbonate skeleton over ice using a Paansche airbrush (Paansche, Inc., Chicago, IL, USA) and homogenate buffer (50 mM phosphate buffer, (pH 7.8) with 0.05 mM dithiothreitol). Four biological replicates were used per treatment (total = 20). Total protein was isolated from crude tissue homogenates (~2 g of coral tissue) as described previously [61 (link)] with the following modifications. Tissue was ground in liquid N₂ with a pre-cooled mortar and pestle in 6 mL of extraction buffer (1.2% β-mercaptoethanol, 0.1 M Tris-HCl (pH 8.8), 10 mM EDTA, 0.9 M sucrose) and 6 mL Tris-saturated phenol (pH 8.8), followed by overnight incubation at room temperature with shaking. All chemicals were obtained from Millipore Sigma (St. Louis, MO, USA) unless noted otherwise. Samples were centrifuged at room temperature for 40 min at 5,000 g. The resulting protein pellets were dissolved in 4 M urea and 0.1% SDS in 10 mM Tris-HCl (pH 8.0). Protein concentration was measured using the EZQ Protein Quantification Kit (Thermo Fisher Scientific, Inc., San Jose, CA, USA) with SoftMax Pro Software v5.3 (Molecular Devices, Inc., San Jose, CA, USA).

MACS-DRG neurons from naïve and day 14 PTX-treated female and male mice were lysed in DiGE lysis buffer (7 M urea, 2 M thiourea, 4% CHAPS, 30 mM Tris-HCl) supplemented with 1× Halt Protease and Phosphatase Inhibitor Cocktail (Thermo). Lysates were quantified with the EZQ Protein Quantification Kit (Thermo). Lysates (20 μg) were added to Bolt bis-Tris 12% gel (Thermo). SDS-PAGE was performed using 1× MOPS SDS Running Buffer (Life Technologies). Proteins were transferred to Immobilon-FL PVDF membrane (Millipore) using 1× Power Blotter 1-Step Transfer Buffer (Invitrogen). PVDF membranes were incubated at 4°C for 48 hours with MHCII and beta tubulin as the loading control, then incubated with 1:2000 donkey anti-rabbit Cy5 secondary antibody for 2 hours at RT. PVDF membranes were imaged using the Typhoon 9600 laser scanner (GE). Band intensities were quantified using AutoQuant imaging software.

EV samples (500 µl aliquots) were thawed on ice and mixed with acetonitrile to a final concentration of 50% (v/v) and evaporated by a centrifugal vacuum concentrator to obtain EV sample proteins for mass spectrometry analysis. The EV sample associated proteins were resuspended in 150 µl of denaturation buffer (7 M urea and 2 M thiourea in 40 mM Tris, pH 8.0).
Protein concentration was measured by EZQ protein quantification kit (Thermo Fisher Scientific), and 30 µg of protein from each sample was reduced with 10 mM dithiothreitol overnight at 4°C. EV protein samples were alkylated the next day with 50 mM iodoacetamide for 2 hours at ambient temperature in the dark and then digested into peptides with 1.2 µg proteomics-grade trypsin/LysC (Promega) according to the SP3 protocol described by Hughes et al. (28 (link)). EV peptides samples were de-salted using ZipTips (Merck) according to the manufacturer’s directions.