Mini protean tgx stain free precast gels 4 20

Protein was mixed with 2 x Laemmli buffer, denatured at 95°C for 10 min, loaded in Mini-PROTEAN TGX Stain-Free Precast Gels 4–20% (Bio-Rad, Hercules, USA), and ran at 120 V for 60 min. Precision Plus Protein Western C (Bio-Rad) was used as a molecular weight standard. The gel was transferred to Bio-Rad Western blot membrane using dry blotting in a Trans-Blot Turbo (Bio-Rad) at 1.3 A and 25 V for 7 min. Blotted membrane was immediately incubated with 5% BSA for one hour at room temperature. Next, the membrane was incubated with primary antibody (anti-penta-his tag) (Qiagen, Hilden, Germany) overnight at 4°C 100 rpm, washed with tris buffered saline with Tween 20 (TBST) three times, incubated with secondary antibodies (Polyclonal rabbit-anti-mouse immunoglobulin/HRP) (Agilent, Santa Clara, USA) for one hour, washed with TBST three times, incubated with HRP substrate Clarity Western ECL (Bio-Rad) for 5 min, and visualized with ChemiDoc XRS+ (Bio-Rad).

HDL particles (1.25 mg/ml) were incubated in the presence or absence of NanA (10 μM), in the presence or absence of PLY (1.7 μM), and in the presence or absence of liposomes and DANA (20 μM) (Carbosynth), in LoBind tubes (Eppendorf, Hamburg, Germany) for 20 min at room temperature. Next, the samples were run through SEC column (Superdex 200 10/300 GL column Global Life Sciences Solutions LLC, Marlborough, MA, United States) at 0.5 ml/min flow rate in PBS. Fractions of 300 μl were collected, and the HDL-2 fractions from 8.3-10.0 ml were pooled and stored immediately at -80 °C until further analysis. The peaks from SEC were run on Mini-PROTEAN TGX Stain-Free precast gels 4-20% in non-reducing conditions (Bio-Rad Laboratories, Hercules, CA) at 120 V, 60 min, stained with silver stain and imaged with Gel Doc XR+ system using silver stain protocol in Image Lab software (Bio-Rad). Protein concentrations of the samples were measured with the Bicinchoninic acid assay (BCA) (Pierce Biotechnology, Rockford, Il, US) according to manufacturer instructions. To estimate the amount of HDL in the assays, the concentrations of HDL samples are expressed as the equivalent concentration (μg/ml) or weight (μg) of protein.

Samples were mixed with 2x SDS buffer mix (Sigma), denatured at 95 °C for 3 min and separated on Mini-PROTEAN^®TGX Stain-Free™ Precast Gels 4–20% (Bio-Rad, Hercules, CA, USA) at 150 volt for 50 min. Precision Plus Protein^™ Western C™ (Bio-Rad) was used as a molecular weight standard. Proteins were transferred to a 0.2 µm Nitrocellulose membrane by dry-blotting in a Trans-Blot^® Turbo™ (Bio-Rad) at 1.3 A, and 25 V for 7 min. Proteins were detected using a BSA-free anti-Penta-His-tag mouse monoclonal IgG (Qiagen, Hilden, Germany) as primary antibody and Polyclonal Rabbit-anti-mouse Immunoglobulin/HRP (Agilent, Santa Clara, CA, USA) as secondary antibody according to the manufacturer´s recommendations. Clarity Western ECL (Bio-Rad) was used as substrate. Visualization of both stain-free SDS-PAGE gel and Western blot was done in a ChemiDoc™ XRS+ (BioRad).