Superblock t20 tbs blocking buffer

Cells were lysed in MPER™ (Mammalian Protein Extraction Reagent, Thermo Scientific, Rockford, IL), which was supplemented with cOmplete Protease Inhibitor Cocktail tablets and PhosSTOP Phosphatase Inhibitor Cocktail tablets (Roche, Indianapolis, IN). Protein concentrations of lysates were determined using the BCA assay (Thermo Scientific, Rockford, IL) and proteins were resolved on SDS-PAGE gels and transferred to PVDF membrane (EMD Millipore, Billerica, MA) using standard procedures. Membranes were blocked with SuperBlock™ T20 (TBS) Blocking Buffer (Thermo Scientific, Rockford, IL) and incubated overnight at 4°C in primary antibodies of Runx2 (Cell Signaling, #8486, 1:1000), ALP (Abcam, #ab67228, 1:1000), RANKL (Cell Signaling, #4816, 1:1000), Osteocalcin (Santa Cruz, #sc-30044, 1:500), Osterix (Abcam, #ab22552, 1:500), and β-actin (Sigma-Aldrich, #A1978, 1:5000). Membranes were then incubated with either anti-mouse (Cell Signaling, #7076, 1:2000) or anti-rabbit IgG (Cell Signaling, #7074, 1:2000), HRP-linked secondary antibodies and Chemiluminescented by Pierce™ ECL Western Blotting Substrate (Thermo Scientific, Rockford, IL). Quantification of signals on Western blots was done using National Institutes of Health (NIH) ImageJ Imaging and Processing Analysis Software with signaling intensity normalized to β-actin.

For assay optimization phosphate-, tris(hydroxymethyl)aminomethan (TRIS)- and triethanolamine (TEA)-based buffers with different concentrations of dimethyl sulfoxide (DMSO) were compared. Furthermore, different reporter molecules, reagent concentrations, detection systems, and the use of detergents (Tween-20, Triton X-100, Triton X-114, IGEPAL, Brij 35, CHAPSO; Sigma-Aldrich) were evaluated. Following these multiple optimization steps, a new standard protocol was defined: MaxiSorp immunoassay plates were coated with 5 μg/ml of mAb JD5.1 overnight at 4°C. After washing the plate twice with PBST, the plate was blocked with SuperBlock T20 (TBS) blocking buffer (Thermo Scientific) for 1 h at 37°C. After another washing step, serial dilutions of the samples in LW buffer (0.2 M TEA, pH 7.5, with 20% DMSO) were added to the plates and incubated in the dark for 2 h at 37°C. Without washing, 100 μL of an 80 ng/ml solution of the reporter molecule MG-161 (Fig 1) in LW buffer was added to the plate and incubated for an additional 45 min. Subsequently, plates were washed four times, and bound MG-161 was detected after 1 h incubation at 37°C by horseradish peroxidase-coupled streptavidin (SouthernBiotech) diluted 1:5000 in PBST. Signal development was done with 3,3’,5,5’-tetramethylbenzidine (TMB) for 5 min after which the reaction was stopped with 0.5 M sulphuric acid.