Maxis 4g cpr

We analyzed the BALF proteome in patients with acute and chronic HP in five cases of each with 2‐DE. The proteins were evaluated as described previously.⁸, ¹³ The gels were scanned using a FluoroPhoreStar 3000 image analysis system (Anatech) and analyzed with Progenesis PG220 Software (Nonlinear Dynamics Ltd.). The proteins were analyzed automatically using the spot detection feature of the software, with automatic warping and matching. Spot volumes were corrected for background and then normalized. The normalized spot volume was calculated as a percentage of the volume of each spot to the volumes of all spots in a gel. Proteins were identified by liquid chromatography nano‐electronspray ionization tandem mass spectrometry (LC‐nESI‐MS/MS) in the Laboratory of Cytometry and Proteome Research at Tokyo Medical and Dental University. LC separation was performed by the nano‐UHPLC system (BrukerDaltonics). Mass analysis was performed on a maXis‐4G‐CPR (BrukerDaltonics) mass spectrometer equipped with a nano‐ESI source.

KLM1 cells were cultured on the hydrogels for 5 days. All cells were removed by incubation with 0.5% trypsin–EDTA for 15 min at 37 ℃, and the protein fragments remaining on the surface of hydrogel were diluted in 100 µl of sample buffer. Complete removal of remaining cells was confirmed by nuclear staining with Hoechst 33258. Then, the samples were separated by one-dimensional sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) using a SuperSep™ Ace, 5–20% gradient gel (Wako). To visualize the proteins bound from the hydrogels, silver staining was performed using a silver staining MS kit (Wako) according to the manufacturer’s protocol. To conduct proteome analysis of lysates, DetergentOUT GBS10 (G-Bioscience, USA) was used to remove SDS from the samples. The concentration of lysates after SDS removal was determined by Nanodrop (Thermo Fisher Scientific, USA). Samples were analyzed by electrospray ionization quadrupole time-of-flight (ESI-QTOF) liquid chromatography-mass spectrometer maXis-4G-CPR (Bruker, Germany). The obtained MS/MS data were searched for the Swiss-Prot database using MASCOT software ver. 2.4.1. for protein identification.