Precellys ck14 kit ceramic 1 4mm

Lung tissue was homogenised (Precellys CK14 (Kit ceramic, 1,4mm, 2ml tubes, #432-3751, settings 5000-2×50, Precellys®24 tissue homogeniser, Bertin Technologies) in MPer (Thermo Fisher Scientific, Paisley, UK), lysed under constant agitation (4°C, 20 minutes) and supernatant collected. Protein content of lysed tissue supernatant was determined by BCA (Pierce®BCA Protein Assay Kit, Thermo Scientific), according to manufacturer’s instructions, to enable equivalent loading. Samples were incubated at 95°C for 10 minutes in sample buffer and reducing agent (NuPage LDS Sample buffer (4x) and NuPage Sample Reducing Agent (10x), Invitrogen). SDS page on 4-12% Bis/Tris gels in MES buffer (Invitrogen) was performed, protein was transferred to nitrocellulose (Nitrocellulose Membrane Filter paper, Sandwich, 0.2μm pore size, Life technologies) and membranes were exposed to anti-mCRAMP antibody (1:500, CRAMP antibody, PA-CPPL-100, Innovagen AB, Lund, Sweden) and anti-β-actin antibody (1:20000, clone AC-15, Sigma-Aldrich), followed by the respective IRDye®680LT or 800CW Li-COR labelled secondary antibody (Li-Cor Biotechnology, Cambridge, UK). Fluorescence intensity was quantified using a Li-Cor BioScience Odyssey 9120 Infrared Imaging System).