Calculate the volumes of the 100 mM PNPP stock solution and buffer (final volume of 1.00 ml) needed to get each of the substrate concentrations.
please fill out the table I need it for lab tomorrow
At the start of the lab, you will be given stock preparations of alkaline phosphatase and PNPP on
ice—keep them that way. You will also be instructed on the volume of enzyme solution to be
added to reaction mixtures. Typically, this lab uses 5 μl of enzyme dilution. Before the start of lab, calculate the
volumes in μl of each component you need to mix together for each of these samples.
For all of the following assays, use the amount of enzyme noted above and vary the substrate
concentration. Use the following assay format and PNPP concentrations of 0.0, 0.20, 0.40, 0.60,
0.80, and 1.0 mM. Calculate the volumes of the 100 mM PNPP stock solution and buffer (final
volume of 1.00 ml) needed to get each of the substrate concentrations. A single blank tube, with
alkaline buffer only, is sufficient, because PNPP has no absorbance at 405 nm.
![**Table 2: Experimental Setup for PNPP Concentration Study**
This table outlines the preparation of different reaction mixtures to study the effect of varying p-nitrophenyl phosphate (PNPP) concentrations on enzyme activity. Each tube contains a unique combination of PNPP, buffer, and enzyme amounts.
- **Blank Tube:**
- **PNPP (µl):** 0
- **Buffer (µl):** 1000
- **Enzyme (µl):** 0
- **Final Volume (µl):** 1000
- **[PNPP] in µM:** 0
- Purpose: Acts as a control to measure background activity without substrate.
- **Tube 1:**
- **PNPP (µl):** 5
- **Buffer (µl):** 1000
- **Enzyme (µl):** 5
- **Final Volume (µl):** 1000
- **[PNPP] in µM:** 200
- **Tube 2:**
- **PNPP (µl):** 10
- **Buffer (µl):** 990
- **Enzyme (µl):** 5
- **Final Volume (µl):** 1000
- **[PNPP] in µM:** 400
- **Tube 3:**
- **PNPP (µl):** 15
- **Buffer (µl):** 985
- **Enzyme (µl):** 5
- **Final Volume (µl):** 1000
- **[PNPP] in µM:** 600
- **Tube 4:**
- **PNPP (µl):** 20
- **Buffer (µl):** 980
- **Enzyme (µl):** 5
- **Final Volume (µl):** 1000
- **[PNPP] in µM:** 800
- **Tube 5:**
- **PNPP (µl):** 25
- **Buffer (µl):** 975
- **Enzyme (µl):** 5
- **Final Volume (µl):** 1000](/v2/_next/image?url=https%3A%2F%2Fcontent.bartleby.com%2Fqna-images%2Fquestion%2Fe31029a8-64b8-4001-b02b-a4ced2e71261%2F49ac6578-b9a7-413c-9dcb-7ba1cd699d78%2F89rsqco_processed.jpeg&w=3840&q=75)
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