Fixed Time Assay 1.

Take 7 clean cuvettes and label them 1-7; to each add 1mL of Z-buffer and 0.2mL of ONPG (4mg/mL) using a 1mL pipette. Note the ONPG is heat and light sensitive so it should be kept in the refrigerator. 2. Using the samples collected from the column (flow through, wash 1, wash 2, wash 3, elution 1, elution 2, elution 3) add 100 microliters of each sample to the Z-buffer/ONPG solution in each cuvette, keeping track of the time at which you added each sample to buffer solution. 3. Once the reaction in a cuvette has gone for one minute, you will stop the reaction by adding 0.5mL of sodium bicarbonate. Pay attention to whether the color turns yellow or not. Yellow indicates the formation of product by the enzyme. The darker the tine, the more concentration of enzyme there is. 4. Check the absorbance spectra at 410nm on the spectrophotometer. 5. Now calculate how much units of beta-galactosidase/mL there is in your samples, using the formula below. (absorbance at 410nm/min) x (Vr) x (D) (Ve) x (Extinction coefficient of ONPG) Units B-gal/mL

Dilution factor should equal 1. Extinction coefficient = Bradford Microassay 1. Warm the spectrophotometer 15 minutes before use. 2. Dilute samples with HisTag binding buffer to an estimated concentration of 1 to 20 g/L. 3. Prepare standard solution (Ovalbumin) with 1mg/mL. 4. Create standards containing a range of 1 to 20 g of protein to a standard volume of 200 L. 5. Prepare unknowns to estimated amounts of 1 to 20 g of protein per tube same volume as the unknowns. 6. Add 800 L of Bradford reagent and incubate 5 minutes. 7. Measure the absorbance at 590nm. 8. Use standard values to create a standard curve. 9. Calculate the amount of unknown protein in each sample tested based upon the standard curve values.