Lesson over Spectrophotometry Assume that you are preparing

(Lesson over Spectrophotometry)

Assume that you are preparing a new dye-based assay in your lab. Nobaody has ever used it before, so you are on your own. Provide a flow-cahrt or a brief list of steps that you would follow to decelop a quantitative assay using this dye.

Solution

1. Prepare a series of protein standards diluted with 0.15 M KCl to final concentrations of 0. ( blank = KCl only), 200,400,600,800,1000 ug/ml. Also prepare serial dilutions of the unknown sample to be measured.

2. Add 100ul of each of the above to the separate test tube ( or spectrophotometer tube if using spec20).

3. Add 5 ml of our own laboratory prepared dye ( say bio X dye) to each tube and mix by vortex or inversion.

4. Adjust the spectrophotometer to a wavelength of 595nm, and blank using the tube which contains no protein.

5. Wait 5 mins and read each of the standards and each of the samples at 595nm wavelength.

6. Plot the absorbance of the standards vs. their concentration. Compute the extinction coefficient and calculate the concentrations of the unknown samples.