2. Label 20 test tubes. Add 50 µl
pNPP of 3, 6, 12, 30, 60 mM to each
tube.
3
6
12
30
60
50 µl
200 µl
Set I,
No inhibitor
0
15
Cit
bu r at e
ff e
r
Substrate
[pNPP]
Add 200 µl Citrate buffer
to set I tubes.
µl
Na3PO4
50 µl
Add 150 µl Citrate buffer
to set II tubes. Add 50 µl
Na3PO4 to set II tubes
Set II,
With inhibitor
20 Test Tubes
Total 250 µl for all tubes.
Vortex to mix thoroughly.
3. Set all reaction tubes and enzyme tube in 37oC water bath.
Incubate for 5 min.
Carry out step 3 by the water bath. See next page for details.
T = 0’, add 250µl Enzyme to assay tube, vortex, back to water
bath. Repeat every 30’ for the following tubes.
T = 10”, add 500µl NaOH to assay tube, vortex, remove from
water bath. Repeat every 30’ for the following tubes.
Back to your bench for step 4.
4. Tubes
En
zy
me
Set I,
No inhibitor
Na
OH
T = 0’, add 250µl Enzyme
to assay tube, vortex, back
to water bath. Repeat every
30’ for the following tubes.
T = 10”, add 500µl NaOH to
assay tube, vortex, remove
from water bath. Repeat every
30’ for the following tubes.
☆ Reaction time for each tube is
Set II,
With inhibitor
3
6
12
30
3’
6’
12’
30’ 60’
3+I
60
6+I 12+I 30+I 60+I
3’+I 6’+I 12’+I 30’+I 60’+I
0’,
2”,
4”,
6”,
8”,
10”,
12”,
14”,
16”,
18”,
30’,
2”30’
4”30’
6”30’
8”30’
10”30’ 12”30’
,
,
1”,
14”30’ 16”30’ 18”30’
,
,
,
,
,
,
3”,
11”,
1”30’
,
5”,
7”,
9”,
13”,
15”,
17”,
19”,
3”30’
5”30’
7”30’
9”30’
5. Add 2 ml d.H2O to each reaction tube. Vortex to mix.
Read OD 405nm.
405nm
ath
ht p
Lig
Sample
1
2
3
4
5
6
7
8
9
10
OD405
0.0451
0.0655
0.1136
0.1082
0.1967
0.2104
0.3898
0.3971
….
….
Make two Lineweaver-Burk plot in one graph. Estimate Vmax and Km in
the presence and absence of inhibitor. Identify what type inhibitor
sodium phosphate is.
6. Add 2 ml d.H2O to each reaction tube. Vortex to mix.
Read OD 405nm.
405nm
ath
ht p
Lig
Sample
1
2
3
4
5
6
7
8
9
10
OD405
0.0451
0.0655
0.1136
0.1082
0.1967
0.2104
0.3898
0.3971
….
….
Make two Lineweaver-Burk plot in one graph. Estimate Vmax and Km in
the presence and absence of inhibitor. Identify what type inhibitor
sodium phosphate is.
![Label 20 test tubes. Add 50 µl
pNPP of 3, 6, 12, 30, 60 mM to each
tube.
3
6
12
30
60
50 µl
200 µl
Set I,
No inhibitor
0
15
Cit
bu r at e
ff e
r
Substrate
[pNPP]
Add 200 µl Citrate buffer
to set I tubes.
µl
Na3PO4
50 µl
Add 150 µl Citrate buffer
to set II tubes. Add 50 µl
Na3PO4 to set II tubes
Set II,
With inhibitor
20 Test Tubes
Total 250 µl for all tubes.
Vortex to mix thoroughly.](data:image/gif;base64,R0lGODlhAQABAIAAAAAAAP///yH5BAEAAAAALAAAAAABAAEAAAIBRAA7)