calculating the “Activity ([PNPP]) for each Supernatent.
Biochemistry
6th Edition
ISBN:9781305577206
Author:Reginald H. Garrett, Charles M. Grisham
Publisher:Reginald H. Garrett, Charles M. Grisham
Chapter18: Glycolysis
Section: Chapter Questions
Problem 8P
Related questions
Question
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
3) For steps 3 through 8, you will use Acid phosphatase assays to determine the amounts of the enzyme in
your supernatants.
Prepare the following dilutions of each supernatant to determine the phosphatase activity (you need at least
0.2 ml final volume for each fraction):
150μ1
Supernatant
I
11
III
IV
V
V
BLANK
Sup 1
Sup 2
Sup 3
Sup 4
V
VI
لبر3000
Reagent
1.0 M Sodium acetate
0.1 M MgCl2
H₂0
Sup 5
Sup 6
SUP 1/3
Dilution
1/100
1/100
1/20
1/100
1/50
1/25
1/50
0
1
2
9
√4) Prepare ready reaction for each fraction, plus a control containing no protein. You can prepare a mix
containing the chemicals below. Because of pipeting errors, prepare a mix for eight tubes (you use seven
tubes). Aliquot 0.3 ml of the mix out to each of the seven tubes. had to defrost sups
500
SOO
min.
3 Min.
4 Min.
Volume Sup
LS
1.5
7.5
1.5
0.05 ml
0.05 ml
2,000 pl 0.20 ml
10
11
12
13
14
Volume/tube
15
16
H₂0
1485
142.5
5) To each of the assay reactions, add 150 uL of either the appropriate diluted fraction (from step 3, one
per tube) or H₂O (as the control). Therefore, six of the tubes will each contain one of the supernatants, the
seventh tube contains no enzyme (water).
6) You will now perform the assays. In order to ensure that each sample incubates for the same amount
of time, samples will be started and stopped in intervals. For example, sample 1 will start at time 0 and
end at time 10, sample 2 will start at time 1 and end at time 11, etc.
For more
activity.
Therefore, at one minute intervals, to each tube successively add 50 uL 10mM PNPP, vortex, and
immediately place into a 30BC water bath.
Supernatent
Starting Time
Ending Time
min.
A-405nm
148.5
47
0.00
0.23
0.41
0.23
0,29
144
147
10.17
0.39
✓8) Read the A405. Use the no protein control as the blank.
Volume in mix
0.4 mL
0.4 mL
1.6 mL
noo
49.5-3
→ 49.5.3
749.5.3
Dilution
Factor
100
100
20
100
50
25
Master Mix
ابر3000
5 min.
6 min.
17 min.
7) Starting at 10 minutes, remove one sample per minute (i.e., exactly 10 minutes for each sample), add 1
ml 1 M NaOH.
Activity
([PNPP])](/v2/_next/image?url=https%3A%2F%2Fcontent.bartleby.com%2Fqna-images%2Fquestion%2F81e33e53-6cee-49a0-88a3-901e9c69b620%2Fcbba939f-a759-494b-a7aa-47270d68aec7%2Fb7z3a49_processed.jpeg&w=3840&q=75)
Transcribed Image Text:Purification Table Part 2
3) For steps 3 through 8, you will use Acid phosphatase assays to determine the amounts of the enzyme in
your supernatants.
Prepare the following dilutions of each supernatant to determine the phosphatase activity (you need at least
0.2 ml final volume for each fraction):
150μ1
Supernatant
I
11
III
IV
V
V
BLANK
Sup 1
Sup 2
Sup 3
Sup 4
V
VI
لبر3000
Reagent
1.0 M Sodium acetate
0.1 M MgCl2
H₂0
Sup 5
Sup 6
SUP 1/3
Dilution
1/100
1/100
1/20
1/100
1/50
1/25
1/50
0
1
2
9
√4) Prepare ready reaction for each fraction, plus a control containing no protein. You can prepare a mix
containing the chemicals below. Because of pipeting errors, prepare a mix for eight tubes (you use seven
tubes). Aliquot 0.3 ml of the mix out to each of the seven tubes. had to defrost sups
500
SOO
min.
3 Min.
4 Min.
Volume Sup
LS
1.5
7.5
1.5
0.05 ml
0.05 ml
2,000 pl 0.20 ml
10
11
12
13
14
Volume/tube
15
16
H₂0
1485
142.5
5) To each of the assay reactions, add 150 uL of either the appropriate diluted fraction (from step 3, one
per tube) or H₂O (as the control). Therefore, six of the tubes will each contain one of the supernatants, the
seventh tube contains no enzyme (water).
6) You will now perform the assays. In order to ensure that each sample incubates for the same amount
of time, samples will be started and stopped in intervals. For example, sample 1 will start at time 0 and
end at time 10, sample 2 will start at time 1 and end at time 11, etc.
For more
activity.
Therefore, at one minute intervals, to each tube successively add 50 uL 10mM PNPP, vortex, and
immediately place into a 30BC water bath.
Supernatent
Starting Time
Ending Time
min.
A-405nm
148.5
47
0.00
0.23
0.41
0.23
0,29
144
147
10.17
0.39
✓8) Read the A405. Use the no protein control as the blank.
Volume in mix
0.4 mL
0.4 mL
1.6 mL
noo
49.5-3
→ 49.5.3
749.5.3
Dilution
Factor
100
100
20
100
50
25
Master Mix
ابر3000
5 min.
6 min.
17 min.
7) Starting at 10 minutes, remove one sample per minute (i.e., exactly 10 minutes for each sample), add 1
ml 1 M NaOH.
Activity
([PNPP])
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