Micro bca protein assay kit

Protein concentration was measured by a MicroBCA Protein Assay Kit (Thermo Scientific, Pierce Biotechnology, USA) using a spectrophotometer (UVM340, ASYS). The Thermo Scientific™ Micro BCA™ Protein Assay Kit is a detergent-compatible bicinchoninic acid formulation for the colorimetric detection and quantitation of total protein. An adaptation of the Thermo Scientific™ BCA Protein Assay Kit (Product No. 23225), the Micro BCA Kit has been optimized for use with dilute protein samples (0.5–20 µg/mL). The unique patented method uses bicinchoninic acid (BCA) as the detection reagent for Cu+1, which is formed when Cu+2 is reduced by protein in an alkaline environment. A purple-colored reaction product is formed by the chelation of two molecules of BCA with one cuprous ion (Cu+1). This water-soluble complex exhibits a strong absorbance at 562 nm that is linear with increasing protein concentrations. The result is an extremely sensitive colorimetric protein assay in a test tube or microplate assay format.

Protein concentration of EV samples from UC and dUC was quantified using the Micro BCA™ Protein Assay Kit (Thermo Fisher Scientific, MA, USA), following manufacturer’s instructions. Protein quantification of EV samples isolated using Exoquick was performed with Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, MA, USA) (following manufacturers’ protocol) since EXO EVs contain high protein concentration, often out of the limit of quantification of Micro BCA™ Protein Assay Kit. Protein was quantified in a FLUOstar Omega microplate reader (BMG LABTEC, Ortenberg, German) at 562 nm.

MMP-12 concentrations in the left L4, L5, and L6, and right L5 spinal nerves, harvested from rats euthanized by carbon dioxide exposure, were determined using an MMP-12 activity assay (AS-71157, AnaSpec, CA, USA). Specimens were homogenized in assay buffer containing 0.1% Triton X-100 and centrifuged at 10000 ×g for 15 min at 4°C. The supernatant obtained was further diluted in assay buffer by 10-fold. MMP-12 concentrations were calculated relative to a human MMP-12 catalytic domain (1 μg/ml, AS-55525, Anaspec, CA, USA) standard curve. 1 mM 5-FAM-Pro-Leu-OH was used as a 5-FAM fluorescence reference standard for instrument calibration. 50 μl of each diluted spinal nerve specimen supernatant, human MMP-12 catalytic domain dilutions, 5-FAM-Pro-Leu-OH dilutions, and a substrate control containing assay buffer only were added to a 96-well plate. 50 μl of MMP-12 substrate was added to each well before incubating at 37°C for 45 min. Thereafter, 50 μl stop solution was added in all wells, and the fluorescence intensity at Ex/Em = 490/520 nm was measured (PHERAstar fluorimeter, BGM Labtech, Germany). Total protein for each sample was measured relative to an albumin standard curve, using a Micro BCA Protein Assay Kit (Micro BCA Protein Assay Kit, Thermo Scientific, MA, USA).