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Questions and Answers
What is the correct pH of the Tris buffer used in this protocol?
What component is specifically mentioned as being light sensitive in this procedure?
How long should you wait for the spectrophotometer's bootup tests to finish?
At what wavelength should the spectrophotometer be set for this assay?
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What is the total volume of the reaction mixture prepared in the microcentrifuge tube labeled 'EA'?
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What is the purpose of inverting the tube several times after mixing the reaction components?
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When preparing to use the spectrophotometer, what should be checked first?
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Which of the following materials is NOT listed as part of the necessary equipment for the Alkaline Phosphatase Assay?
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What should you do first when using the spectrophotometer?
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What is the consequence of pressing the blank button again after initial blanking?
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How often should you take absorbance readings after the initial measurement?
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What is the purpose of mixing the alkaline phosphatase solution in the cuvette?
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What safety measure should be taken after completing the experiment?
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What is essential to confirm before adding the enzyme to the spectrophotometer?
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Why is it important to proceed quickly after mixing the cuvette?
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What should you do to ensure data accuracy when recording absorbance measurements?
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Study Notes
Alkaline Phosphatase Assay Protocol
- This protocol is used to measure the activity of alkaline phosphatase (ALP) using a spectrophotometer.
- ALP catalyzes the hydrolysis of p-nitrophenyl phosphate (pNPP) to p-nitrophenol (pNP), which absorbs light at 405nm.
Materials
- Spectrophotometer with cuvette function
- Plastic cuvette
- 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
Preparation
- Prepare a table in your lab notebook with columns for Time (min) and Absorbance.
- Record time points at 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 minutes.
Enzyme Assay
- Prepare a reaction mixture in a 1.5 ml microcentrifuge tube labeled “EA” for enzyme assay.
- The reaction mixture contains 480 μL of molecular grade water, 500 μL of 0.2 M Tris buffer, pH 8.0, and 10 μL of 1 mM pNPP solution.
- Mix the reaction mixture by inverting the tube several times with the cap closed.
- Protect the reaction mixture from light by covering the tube with aluminum foil.
Spectrophotometer Setup
- Turn on the spectrophotometer and select the cuvette function.
- Select a fixed wavelength of 405nm.
- Prepare a timer to start counting up from 0.
- Place the prepared cuvette containing the reaction mixture into the spectrophotometer and blank it at 405nm.
- This step removes the baseline absorbance of pNPP from the other readings, making all subsequent readings relative to this blank.
Enzyme Addition
- Remove the cuvette from the spectrophotometer.
- Add 10 μL of 2 mg/mL alkaline phosphatase solution to the cuvette.
- Gently mix the solution by pipetting up and down 2-3 times.
- Place the cuvette back into the spectrophotometer and record the first absorbance measurement at t=0.
Data Collection
- Record absorbance readings at 30 sec intervals for 4 min., then at 1 min intervals for 6 min., and take a final reading at 12 min.
- A total of 16 absorbance measurements should be recorded.
- Enter the data into a spreadsheet for analysis.
Cleaning Up
- Dispose of the cuvette and tubes as instructed.
- Empty the ice bucket and leave it upside down on a drying rack or upright by the sink.
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Description
This quiz covers the protocols for measuring alkaline phosphatase (ALP) activity using a spectrophotometer. It includes information on materials required, preparation steps, and the enzyme assay process. You'll learn about the reaction conditions and how to record your results effectively.
