Zentrifuge z 36 hk centrifuge

Serum samples were diluted 5-fold with sodium phosphate buffer (phosphate-buffered saline—PBS), centrifuged at 16,000 rpm for 1 min in a Zentrifuge Z 36 HK centrifuge (Hermle Labortechnik Gmbh, Wehingen, Germany) at 4 °C, and filtered through a standard filter Filtropur S (Sarstedt, Nümbrecht, Germany) with a diameter of 22 microns. The resulting supernatant was passed through a Multiple Affinity Removal Column Human 14 (4.6 × 100 mm), Agilent, Santa Clara, CA, USA) for affinity binding and removal of 14 major proteins: albumin, IgG, IgA, transferrin, haptoglobin, antitrypsin, fibrinogen, alpha2-macroglobulin, alpha1-acid glycoprotein, IgM, apolipoprotein AI, apolipoprotein AII, complement C3, and transthyretin using an Agilent 1200 series HPLC system.
The resulting samples were concentrated via ultrafiltration through 3 kDa Microcon^® centrifuge ultrafilters (Millipore, Molsheim, France) at 14,000 rcf for 15 min at 20 °C. The protein concentration was measured based on the absorbance at 280/260 nm using a Varioskan LUX spectrophotometer (Thermo Scientific, Waltham, MA, USA) located at the core facility Medical Genomics at Tomsk National Research Center.

Fasting venous blood was collected in the morning in sample tubes (Becton Dickinson Vacutainer, Nederland) containing clot formation activator. Serum was isolated from blood by centrifugation for 20 min at 2000×g using the Digicen 21R centrifuge (Orto Alresa, Spain). Then serum was aliquoted and stored at − 80 °C.
Six major high-abundance proteins – serum albumin, IgG, IgА, antitrypsin, transferrin, and haptoglobin were depleted from the sera before the further studies. For this, the samples were five-fold diluted with PBS, centrifuged (4000 g, 4 °C, 5 min) using Zentrifuge Z 36 HK centrifuge (Hermle labortechnik Gmbh, Germany), filtered through Filtropur S 0.2 membrane (Sarstedt), and the supernatant was then passed through the Multiple Affinity Removal Column Human 6 (4.6 × 100 mm, Agilent, USA) for affinity binding of the major proteins, with subsequent concentration of the column flow-through by ultrafiltration through 5 kDa Microcon® Centrifugal Ultrafilters (Millipore, France) in accordance with the protocol provided by the manufacturer. Protein concentration was measured by absorbance at 280/260 nm using Epoch microplate spectrophotometer (BioTek, USA) with the software installed.