2d quant kit

To estimate the number of CHIKV particles per mL in the UV-inactivated extract, we first determined the mass of E2 protein in our preparation as follows. Three volumes: 10, 5 and 2 µL of the CHIKV stock at a total protein concentration of 691 µg/mL 2D-Quant Kit (Sigma Aldrich) were loaded into an SDS-PAGE gel and ran side-by-side with a standard of known mass (0.5 µg of BSA). The density of the E2 bands was quantified by comparison with the density of the BSA standard. The concentration of E2 was estimated in 36 µg/mL, which is 5.15% of the CHIKV UV-inactivated stock. The number of CHIKV particles per mL (CHIKV/mL) in 40 µg/mL of our CHIKV preparation was calculated in 1.2 × 10¹⁰ CHIKV/mL as follows: # of molecules of CHIKV/mL = {[(40 µg/mL ×  0.0515)/ 240)] / 46,000 g/mol} ×   6.022 × 10²³ number of particles/mol x 10^− 6; where 40 µg/mL is the concentration of the CHIKV UV-inactivated in the starting dilution (see above); 0.0515 is the fraction of E2 protein; 46,000 g/mol is the molecular weight of E2; 240 is number of E2 copies per CHIKV particle [28 (link)]; 6.022 × 10²³ number of particles/mol is Avogadro’s number; and 10^− 6 is a factor to convert µmol into mol.

Whole proteins were extracted from all groups of the tumor by protein extraction buffer (0.1 M phenylmethylsulfonyl fluoride (PMSF), 2% β-mercaptoethanol). After vortex and centrifuge, the supernatant transfers to a new tube and add 0.1 M ammonium acetate. Re-moved the supernatant, and add 80% cold-acetone for washing, and did speed-vacuum to dry the pellet. The pellet dissolved in lysis buffer (7 M urea, 2 M thiourea, 4% CHAPS, 1 mM PMSF, and 50 mM dithiothreitol (DTT)). The total soluble protein concentration was determined by the 2-D Quant kit (Sigma-Aldrich, St. Louis, MO, USA) in accordance with the manufacturer’s protocol.

Cartilage and bone were carefully separated using a cutter and a hammer and immediately placed in liquid nitrogen. Frozen cartilage (200 mg) and bone (150 mg) samples were crushed in liquid nitrogen using a hammer and a stainless steel mortar and pestle and then ground to a fine powder in liquid nitrogen using a porcelain mortar and pestle. Protein extraction and quantification(2-D Quant Kit, Sigma Life Science), two dimensional electrophoresis (2-DE) and spot identification were then performed as previously described
[18 (link)]. Spot volume normalization was performed using the Progenesis “Total Volume Ratio” option. Descriptive statistics were done using R scripts and the ade4 package. Principal component analysis (dudi.pca) and between-class correspondence analysis (dudi.bca) were performed to investigate differences between OC-free and OC(D)-affected horses as well as heterogeneity between affected samples. Spots having inertia above the third quartile and showing at least a 50% change in abundance between OC(D)-affected foals and OC-free were considered as associated with the pathology. A hierarchical clustering (Pearson correlation) was also performed based on a subset of differentially expressed spots (t-test, adjusted p-value <0.2).