gloved finger, mix by vortexing (inverting if necessary). You may now place the tube back in your test tube rack on your bench. 13. Wipe your “blank” tube (tube 1) with a kimwipe and use it to blank your spectrophotometer . 14. Measure the absorbance of the solutions in the remaining tubes (make sure to wipe the tubes with a kimwipe and methanol before taking a reading), and record these values in Table 10 below . Convert your absorbance reading to [pNP] ( ! M) using your standard curve line values. Report the [pNP] ( ! M) for each tube to your Lab Instructor . Your Lab Instructor will post this information to the Course canvas site for all students in your section to use for their Post-lab Homework assignment. Table 10. Experiment II. The Enzyme Inhibition Experiment: Fluoride Absorbance Reading Tube 1 2 3 4 5 6 7 Final [pNPP] ( ! M) Absorbance [pNP] ( ! M) 15. NaF WASTE CANNOT BE DISCARDED IN THE SINK . Discard the solution in the test tubes into the NaF liquid waste container located on the center lab bench. Discard materials in their proper receptacle . Tube Time to add 1ml of Na 2 CO 3 1 3 min 2 2.5 min 3 2 min 4 1.5 min 5 1 min 6 0.5 min 7 0 min
3-19 Experiment III. The Enzyme Inhibition Experiment: Sorenson’s Phosphate Materials needed per group • Bottle of pNPP stock (4mM) • Bottle of water • Bottle of pH5 buffer • Bottle of Sorenson’s phosphate (2mM) • Bottle of Enzyme stock (5 ! M) • Bottle of Na 2 CO 3 stock (1M) • Cuvettes and cuvette racks • Kimwipes • Markers • Serological pipets and pipet-aids • Timers • Vortexer • Spectrophotometer • Water bath set to 37°C • Liquid waste beaker 1. Make sure your spectrophotometer is turned on and that the wavelength is set to 420 nm. 2. Obtain 7 glass test tubes and label them 1-7 with a sharpie marker . Be sure to write the labels above the light path of the spectrophotometer. 3. First, make serial dilutions of the substrate (pNPP) according to the Table 11 below. Start with tube 7 and work your way backwards to tube 2 . Make sure to mix well by vortexing (cap the tube with your gloved finger & mix well, inverting if necessary). Use a new pipet for each dilution. Table 11. Experiment III. The Enzyme Inhibition Experiment: Sorenson’s Phosphate Experimental Set-Up Tube Volume H 2 O (ml) Volume 4 mM pNPP stock OR an appropriate NPP dilution from tubes 3-7 (ml) [pNPP] ( ! M) in dilution Final [pNPP] ( ! M) after adding all reagents (except Na 2 CO 3 ) 1 1 ml 0 ml 0 mM 2 1 ml 1 ml from tube #3 125 uM 3 1 ml 1 ml from tube #4 250 uM 4 1 ml 1 ml from tube #5 500 uM 5 1 ml 1 ml from tube #6 1000 uM 6 1 ml 1 ml from tube #7 2000 uM 7 2 mL of 4 mM (4000 uM) pNPP stock 4000 uM
3-20 4. At the end of setting up the dilution series, discard 1 ml from tube 2 into the liquid waste beaker on your lab bench so that you now have 1 ml of solution in each tube.
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- Fall '19
- Enzyme, Enzyme inhibitor, Lab Instructor, PNP, pNPP