mL of Coomassie Blue to each tube and mix by vortex, or inversion. Adjust the spectrophotometer to a wavelength of 595 nm, using the tube which contains no protein (blank). Wait 5 minutes and read each of the standards and each of the samples at 595 nm wavelength. Plot the absorbance of the standards vs. their concentration. Compute the extinction coefficient and calculate the concentrations of the unknown samples. Procedure (Micro Assay, 1-10 µg protein/mL) Prepare standard concentrations of protein of 1, 5, 7.5 and 10 µg/mL. Prepare a blank of NaCl only. Prepare a series of sample dilutions. Add 100 µL of each of the above to separate tubes (use microcentrifuge tubes) and add 1.0 mL of Coomassie Blue to each tube. Turn on and adjust a spectrophotometer to a wavelength of 595 nm, and blank the spectrophotometer using 1.5 mL cuvettes or use a mobile smartphone camera (RGBradford method). Wait 2 minutes and read the absorbance of each standard and sample at 595 nm. Plot the absorbance of the standards vs. their concentration. Compute the extinction coefficient and calculate the concentrations of the unknown samples. Using data obtained to find concentration of unknown In summary, in order to find a standard curve, one must use varying concentrations of BSA (Bovine Serum Albumin) in order to create a standard curve with concentration plotted on the x-axis and absorbance plotted on the y-axis. Only a narrow concentration of BSA is used (2-10 ug/mL) in order to create an accurate standard curve. Using a broad range of protein concentration will make it harder to determine the concentration of the unknown protein. This standard curve is then used to determine the concentration of the unknown protein. The following elaborates on how one goes from the standard curve to the concentration of the unknown. First, add a line of best fit, or Linear regression and display the equation on the chart. Ideally, the R2 value will be as close to 1 as possible. R represents the sum of the square values of the fit subtracted from each data point. Therefore, if R2 is much less than one, consider redoing the experiment to get one with more reliable data. The equation displayed on the chart gives a means for calculating the absorbance and therefore concentration of the unknown samples. In Graph 1, x is concentration and y is absorbance, so one must rearrange the