Protein refolding was performed by diluting the 6 mol/L GdnHCl-denatured proteins in buffer A with a dilution ratio of 1 100.
The best conditions were obtained for the ELISA when the antigen concentration was 0.15 μg/ml carbonate bicarbonate buffer, with non-fat dry milk used for blocking and in the sample buffer, with a serum dilution of 1 100 and the conjugate diluted 1 5000.
To reduce non-specific background, the supernatants were diluted 10-fold with a dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl, pH 8.0, 167 mM NaCl) containing protease inhibitors and Protein A Agarose/Salmon Sperm DNA (Upstate/Millipore Corp).
and diluted 10 times with a dilution buffer (50 m m Tris HCl, pH 8.0, 1 m m EDTA, 150 m m NaCl, 1 m m DTT and 1% Triton X-100), which was supplemented with 1× protease inhibitor cocktail (Nacalai, Japan) and RNase inhibitor (Toyobo).
Stock Au NP solution in 20 mM Tris-HCl buffer (TH, pH 8.0) was prepared by centrifugation of raw Au NP-citrate solution, aspiration of the supernatant, and redispersion in TH buffer with 20× dilution to give a working concentration of 0.64 nM.
Next, they were scraped from the bottom of the dishes with a dilution buffer and then moved to conical tubes.
After fixation/permeabilization cells were washed with 0.5 ml of 1x BD Perm/Wash buffer (BD Biosciences; alternatively with PBS-BSA) and resuspended in 50 μl of 1x BD Perm/Wash buffer with the appropriate dilution of primary antibodies.
Briefly, after incubation with blocking buffer, a serial dilution of the standard as well as a 20,000-fold dilution of serum samples was incubated.
Blot washed two times in TBST buffer, primary antibodies incubated with a 1 1000 dilution of anti-Myc or 1 4000 dilution of anti-FLAG antibodies in TBST for overnight at 4°C.
Following an overnight incubation in blocking buffer, cells were treated with a 1∶1000 dilution (1∶100 for pericentrin) of primary antibody to blocking buffer for 1 hour.
