Amido black staining solution

Proteins were resolved on a 12% SDS-PAGE gel and transferred onto a nitrocellulose membrane (Porablot NCP, Macherey-Nagel). Following blocking with 5% non-fat milk in TBST (0.01 M Tris-HCl pH 8.0, 150 mM NaCl, 0.05% Tween 20), the membrane was sequentially incubated with the primary and HRP-conjugated secondary antibodies diluted in blocking buffer. Proteins were detected using chemiluminescent substrates (Amersham™ ECL™ Select Western Blotting Detection Reagent). Images were obtained with a BioSpectrum 810 imaging system (UVP). For an estimation of the amount of protein loaded, membranes were either probed with an antibody against Gapdh or stained with amido black staining solution (Sigma-Aldrich) after development, according to the manufacturer's instructions.

Frozen renal tissue was homogenized, protein samples were prepared as described [57 (link)] and separated on a denaturing SDS-PAGE gel [58 (link)]. After electrophoresis, the gels were electroblotted onto PVDF membranes (Hybond-P, GE Amersham, Munich, Germany), blocked with Rotiblock (Roth, Karlsruhe, Germany) for 1 h and incubated overnight with a primary antibody to chemerin. Protein bands were visualized with secondary horseradish peroxidase-conjugated IgG antibodies (Santa Cruz Biotechnology, 1:50,000), using the Pierce ECL+ system (Thermo Fisher Scientific, Waltham, MA, USA). Blots were quantified using a luminescent imager (LAS-1000, Fujifilm, Berlin, Germany) and Aida 2.1 image analysis software (Raytest, Berlin, Germany). Loading of the blot was quantified by Amido Black staining solution (Sigma, Taufkirchen, Germany).