BCH 219L Alkaline Phosphatase Assay PDF

BCH 219L Alkaline Phosphatase Assay – Spectrophotometer This protocol was originally written for a standard spectrophotometer (Gene McDonald, BCH 369L). It has been adapted by Amanda Vines Garrett for BCH 219L. Last updated: Spring 2024 Instructions: Work as a table. Make sure you coordinate with o...

BCH 219L Alkaline Phosphatase Assay – Spectrophotometer This protocol was originally written for a standard spectrophotometer (Gene McDonald, BCH 369L). It has been adapted by Amanda Vines Garrett for BCH 219L. Last updated: Spring 2024 Instructions: Work as a table. Make sure you coordinate with others and/or your instructor/UGCA as to when you will use the spectrophotometer so there is not a bottleneck! Also make sure that everyone is involved with the process, even if they are just watching sometimes. Take turns pipetting/using the spectrophotometer. Everyone should be able to repeat the protocol if asked! Materials: Spectrophotometer with cuvette function Plastic cuvette (for visible light) Molecular grade water 0.2 M Tris buffer, pH 8.0 1 mM pNPP 2 mg/mL alkaline phosphatase in Tris buffer P20, P1000 pipettes and tips Sterile microcentrifuge tubes and rack Thin permanent marker Ice bucket Timer Protocol: 1. Prepare your lab notebook to record data. Make a table of Time (min) in one column next to Absorbance in another column. Use the following time points: 0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 12.0. This equates to 30 sec. intervals for 4 min., then 1 min. intervals for 6 more min., and a final reading at 12 min. 2. Set up the following reaction in a 1.5 ml microfuge tube (label “EA” for enzyme assay): Molecular grade water Tris buffer, pH 8.0 pNPP solution 480 μL 500 μL 10 μL 3. Mix by inverting tube several times (with cap closed). If waiting for another group to use the spectrophotometer, pause here. Cover with aluminum foil; pNPP is light sensitive. STOP: Do not proceed without confirmation from your instructor/UGCA that it’s your turn to use the spectrophotometer. 1 4. Pipette reaction mixture into a cuvette. If you’re the first group to use the spectrophotometer today, continue to step 5 along with your UGCA’s assistance. Otherwise, skip to step 6. 5. Turn on the spectrophotometer if it’s not already on (plug in, switch at back). Depending on which spectrophotometer you are using, do one of the following: Go to Live Display (in the middle with a rainbow symbol). Wait for bootup tests to finish (~3-5 min). Make sure that Fixed Wavelength is selected. 6. Make sure that 405nm is selected. Also at this point, make sure you have a timer ready to start counting up from 0. 7. Place prepared cuvette into spectrophotometer (which should still only have pNPP, buffer and water) and blank at 405 nm. This will remove the baseline pNPP absorbance from the other readings automatically, so everything will be measured relative to this one measurement. Make sure you confirm which type of spectrophotometer you are using below BEFORE you add enzyme! You may need to press a button for each new absorbance measurement. For Live Display spectrophotometer: The spectrophotometer will automatically take readings from now on every few seconds. Do NOT press the blank button again!! Otherwise, it will be reset to keep the current reading as a blank and the rest of the absorbance readings will be wrong. For Fixed Wavelength spectrophotometer: The spectrophotometer will NOT take a new reading unless you press Read, so you’ll need to do this for each new timepoint. Do NOT press the blank button again!! Otherwise, it will be reset to keep the current reading as a blank and the rest of the absorbance readings will be wrong. 8. Note: this step is time sensitive. Proceed as quickly as you can without spilling/introducing bubbles to obtain the best data. Have someone ready to start the timer as soon as the cuvette is mixed and returned to the spectrophotometer. Remove cuvette and add 10 μL alkaline phosphatase solution. Using the P1000 pipettor, pipet solution up and down gently 2-3 times to mix. Place cuvette back into spectrophotometer and record the first absorbance measurement as t = 0. 9. Read and record A405 on the spectrophotometer at 30 sec. intervals for 4 min., then at 1 min. intervals for 6 more min., then take a final reading at 12 min. You should have a total of 16 measurements to fill up your table. 10. You’ll also need a digital version of your data for analysis. Start a spreadsheet on your computer and type in your data from the table. Have someone else check your typing to prevent mistakes. (Note: you can just have one person in your group do this and then send to everyone else. However, everyone should also have the raw data in their own lab notebook!) Clean up! 1. Dump the cuvette down the sink and throw away. 2. Throw away your tubes, except ones the UGCAs designate should be kept for next lab. 2  3. If you are the last group using the ice bucket at your table, dump it in the sink and leave upside down on a drying rack or leave upright by sink. 4. Make sure both you and your lab partner(s) have the digital data before you leave lab. Don’t forget data analysis after lab! Details below. Data Analysis: Perform After Lab Your data consists of the blanked absorbance relative to time. Before proceeding, subtract each of the time point absorbances from the one at the “0” time point (the first absorbance reading). This is important since we only want to measure the product formed as a result of enzyme activity over time. 1. Calculate product concentration [P] in mol/L at each time point using the Beer-Lambert equation. ε405 = 1.8 x 104 M-1 cm-1 for p-nitrophenol. The path length for the spectrophotometer is 1 cm. 2. Plot [P] vs. time in sec, and determine initial velocity (V0) of the reaction. (Hint: can you use your whole graph to calculate this, or only some of the data? Which data will be most relevant? What units will velocity be in?) 3