calculating the “Activity ([PNPP]) for each Supernatent.

Biochemistry
9th Edition
ISBN:9781319114671
Author:Lubert Stryer, Jeremy M. Berg, John L. Tymoczko, Gregory J. Gatto Jr.
Publisher:Lubert Stryer, Jeremy M. Berg, John L. Tymoczko, Gregory J. Gatto Jr.
Chapter1: Biochemistry: An Evolving Science
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I need help calculating the “Activity ([PNPP]) for each Supernatent. I need to know how to solve this so I can fill out my purification table worksheet. Please help. This class is Biochemistry 1 Lab.
**Purification Table Part 2**

**Overview:**

For steps 3 through 8, you will use Acid Phosphatase assays to determine the amounts of the enzyme in your supernatants.

---

**Preparation and Dilutions:**

Prepare the following dilutions of each supernatant to determine the phosphatase activity:

| Supernatant | Dilution | Volume Sup (µL) | H2O (µL) | Total Volume (µL) |
|-------------|----------|------------------|----------|------------------|
| V           | 1/100    | 1.5              | 498.5    | 500              |
| VI          | 1/200    | 7.5              | 142.5    | 150              |
| IV          | 1/200    | 7.5              | 142.5    | 150              |
| II          | 1/100    | 7.5              | 142.5    | 150              |
| I           | 1/25     | 9                | 441      | 450              |
| VI (blank)  | 1/750    | 9                | 141      | 450              |

*Note*: You need at least 0.2 ml final volume for each fraction.

---

**Assay Reaction Preparation:**

1. Prepare the assay reaction for each fraction, plus a control containing no protein.
2. Create a mix containing the chemicals below. Due to pipeting errors, prepare a mix for eight tubes but use seven tubes.
3. Aliquot 0.3 ml of the mix into each of the seven tubes (add closest Sups).

*Master Mix Total Volume: 3000 µL*

| Reagent          | Volume/tube (mL) | Volume in mix (mL) |
|------------------|------------------|--------------------|
| 1.0 M Sodium acetate | 0.05             | 0.4                |
| 1.0 M MgCl2          | 0.05             | 0.4                |
| H2O                  | 0.20             | 1.6                |

---

**Assay Execution:**

5. To each of the assay reactions, add 150 µL of either the appropriate diluted fraction (from step 3, one per tube) or H
Transcribed Image Text:**Purification Table Part 2** **Overview:** For steps 3 through 8, you will use Acid Phosphatase assays to determine the amounts of the enzyme in your supernatants. --- **Preparation and Dilutions:** Prepare the following dilutions of each supernatant to determine the phosphatase activity: | Supernatant | Dilution | Volume Sup (µL) | H2O (µL) | Total Volume (µL) | |-------------|----------|------------------|----------|------------------| | V | 1/100 | 1.5 | 498.5 | 500 | | VI | 1/200 | 7.5 | 142.5 | 150 | | IV | 1/200 | 7.5 | 142.5 | 150 | | II | 1/100 | 7.5 | 142.5 | 150 | | I | 1/25 | 9 | 441 | 450 | | VI (blank) | 1/750 | 9 | 141 | 450 | *Note*: You need at least 0.2 ml final volume for each fraction. --- **Assay Reaction Preparation:** 1. Prepare the assay reaction for each fraction, plus a control containing no protein. 2. Create a mix containing the chemicals below. Due to pipeting errors, prepare a mix for eight tubes but use seven tubes. 3. Aliquot 0.3 ml of the mix into each of the seven tubes (add closest Sups). *Master Mix Total Volume: 3000 µL* | Reagent | Volume/tube (mL) | Volume in mix (mL) | |------------------|------------------|--------------------| | 1.0 M Sodium acetate | 0.05 | 0.4 | | 1.0 M MgCl2 | 0.05 | 0.4 | | H2O | 0.20 | 1.6 | --- **Assay Execution:** 5. To each of the assay reactions, add 150 µL of either the appropriate diluted fraction (from step 3, one per tube) or H
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