A Standard Curve Developed From The Cytoplasmic Protein...

Figure 1 shows a standard curve developed from the cytoplasmic protein standards of lung cancer cells. A gradual increase in corrected absorbance can be notes, which more noticeably increases after 125 ÃŽ ¼g/mL. After this point the values nearly double. The absorbance readings for the samples were recorded along with the standard concentrations [500 ÃŽ ¼g/mL, 250 ÃŽ ¼g/mL, 125 ÃŽ ¼g/mL, 62.5 ÃŽ ¼g/mL, and 0 ÃŽ ¼g/mL (PBS only) †¨The corrected absorbance for the protein standards were found by subtracting the average absorbance of the PBS blanks (0 ÃŽ ¼g of protein) from all other averages. †¨Excel was then used to create a standard curve for the above protein standards, where protein concentration was plotted on the X-axis and corrected absorbances on the y-axis. The linear trend line was used to determine the protein concentration of the diluted sample according to the average corrected absorbance values. The dilution factor was accounted for by multiplying the calculated concentrations of all protein samples by 20. The volume (in ÃŽ ¼L) needed to load an equal amount of the volume (in ÃŽ ¼g) of the second protein sample was calculated, followed by the calculation of the RIPA volume needed to fill the protein sample to 10 ÃŽ ¼L. Four times the volume was then calculated for each volume and these values were used for the SDS-PAGE procedure the following week. The sample with the lowest concentration was used to calculate the amount of protein (in ÃŽ ¼g) used in the 10 ÃŽ ¼L solution, indicating the maximum