SimpleStep ELISA technology allows the formation of the antibody-antigen complex in one single step, reducing assay time to 90 minutes. Add samples or standards and antibody mix to wells all at once, incubate, wash, and add your final substrate. See protocol for a detailed step-by-step guide.

Example of human Cytochrome C standard curve in 1X Cell Extraction Buffer PTR + Enhancer.

Interpolated concentrations of native Cytochrome C in human heart tissue extract based on a 20 μg/mL extract load, colon tissue extract based on a 50 μg/mL extract load, and skeletal muscle tissue extract based on a 25 μg/mL extract load. The concentrations of Cytochrome C were measured in duplicate and interpolated from the Cytochrome C standard curve and corrected for sample dilution. The interpolated dilution factor corrected values are plotted (mean +/- SD, n=2). The mean Cytochrome C concentration was determined to be 35.41 ng/mL in heart tissue extract, 32.74 ng/mL in colon tissue extract, and 43.12 ng/mL in skeletal muscle tissue extract.

Other species reactivity was determined by measuring a 20 g/mL extract load of human and rat heart tissue extract samples, interpolating the protein concentrations from the human standard curve, and expressing the interpolated concentrations as a percentage of the protein concentration in the human heart tissue extract. Cross-reactivity was determined to be 100% in rat heart extract. Due to 100% amino acids sequence identity of rat and mouse Cytochrome C, the same cross-reactivity can be assumed for mouse Cytochrome C.

Comparison Cytochrome C distribution in subcellular fractions derived from 3.7x103 HeLa cells and whole cells cultured in the presence (treated) or absence (untreated) of 1 µM staurosporine for 4 hours. Cells were collected directly after treatment and subcellular fractions were prepared using a cell fractionation kit (ab109719). Fractions were processed as described in section 11.10. and assayed. The concentrations of Cytochrome C were measured in three different dilutions of the fraction samples in duplicates and interpolated from the Cytochrome C standard curve. The interpolated values are plotted (mean +/- SD, n=3). The mean Cytochrome C concentration was determined be 171.4 ng/mL in the treated cytosol fraction, 242.8 in the treated mitochondrial fraction, 562.0 in the treated whole cell sample, 407.2 ng/mL in the untreated mitochondrial fraction, and 558.9 ng/mL in the untreated whole cell sample. Cytochrome C was not detectable in the untreated cytosol fraction and in both nuclear fractions.

Interpolated concentrations of native Cytochrome C in human extract samples. The concentrations of Cytochrome C were measured in three different dilutions in duplicate and interpolated from the Cytochrome C standard curve and corrected for sample dilution. The interpolated dilution factor corrected values are plotted in ng of Cytochrome C per mg of extract (mean +/- SD, n=3). Cytochrome C concentration was determined to be 1716 ng/mg heart tissue extract, 670.1 ng/mg in colon tissue extract, 1689 ng/mg in skeletal muscle tissue extract, 924.2 ng/mg in HepG2 cell extract, 1658 ng/mg in PC-3 cell extract, and 386.2 ng/mg in THP-1 cell extract samples.

Interpolated concentrations of native Cytochrome C in HepG2 cell extract, PC-3 cell extract, and THP-1 cell extract samples based on a 50 μg/mL extract load. The concentrations of Cytochrome C were measured in duplicate and interpolated from the Cytochrome C standard curve and corrected for sample dilution. The interpolated dilution factor corrected values are plotted (mean +/- SD, n=2). The mean Cytochrome C concentration was determined to be 45.64 ng/mL in HepG2 cell extract, 81.23 ng/mL in PC-3 cell extract, and 19.01 ng/mL in THP-1 cell extract.