Editing Purification of Tth-Muts (section)

== ATPase assay ==
<pre>TEA  buffer:                    2L/1L of 1X:       50 mL of 10X:
50 mM triethanolamine-HCl (TEA) 18.57 g/9.29 g     4.64 g
50 mM KCl                       7.46 g/3.73 g      1.86 g
20 mM MgCl2*6H2O                8.13 g/ 4.07 g     2.03 g
pH 7.5
</pre>

'''ATP:,100 mM, in H2O'''

5 mL: 0.275 g

pH 7.5 (pH changes quickly!)



'''Ammonium molybdate: 4.2%, in 4 N HCl 50 mL'''

2.1 g (NH4)6Mo7O24

16.51 mL of 12.1 N HCl



'''H3PO4, 1000 µM, in 1 N H2SO4'''

50 mL: 58 µL of 85% H3PO4

1.38 mL of 36 N H2SO4

Add H2O to 50 mL

'''Citric acid 34%, in H2O'''

50 mL: 17 g

H2SO4, 0.01 N, in H2O

50 mL: 138 µL of 36 N



'''Triton X-100, 1%, in H2O'''

50 mL: 0.5 mL of 100%

'''Malachite green 0.045%, in H2O'''

36 mL: 16.2 mg

Make dye solution (make day of experiment, make fresh every time):

*Combine 36 mL of 0.045% malachite green, 12 mL of 4.2% ammonium molybdate, and 1 mL of 1% Triton X-100
*Let dye solution sit on bench top at least 1 hour
*Filter through 0.45 µm filter (with dye recipe as written, can do one PO4 standard curve and seven protein combinations)
*After filtering, let dye solution sit on bench top 1 hour before using

Phosphate Std

{|
|-
| 
Final PO4 conc (uM)

| 
uL of 1000 µM H3PO4 (in 1 N H2SO4)

| 
uL of 0.01 N H2SO4

|-
| 
0

| 
0

| 
1000

|-
| 
20

| 
20

| 
980

|-
| 
40

| 
40

| 
960

|-
| 
60

| 
60

| 
940

|-
| 
80

| 
80

| 
920

|-
| 
100

| 
100

| 
900

|-
| 
120

| 
120

| 
880

|-
| 
140

| 
140

| 
860

|-
| 
160

| 
160

| 
840

|-
| 
180

| 
180

| 
820

|-
| 
200

| 
200

| 
800

|}

'''Perform phosphate standard curve:'''

a. Blank spec at 660 nm with 1 mL H2O

b. Combine 800 µL dye solution and 50 µL of 1X TEA in eleven 1.5 mL tubes

c. Add 50 µL of phosphate standard solution, start timer, vortex, incubate 60 sec

d. At 1 minute, add 100 µL of 34% citric acid, vortex, pipette into cuvette

e. Take three readings at 660 nm at 1 min 25 sec, 1 min 30 sec, 1 min 35 sec

f. Pour liquid from cuvette into waste container

g. Repeat steps c-f for all standards

'''Perform steady-state ATPase assay with proteins''':

a. Thaw ATP aliquot, keep at room temp

b. Combine:

i. 255 µL H2O

ii. 37.5 µL of 10X TEA buffer

iii. 37.5 µL of 1.25 µM protein (final 0.125 µM)

iv. 37.5 µL of 3 µM protein (final 0.3 µM) or 37.5 µL of 1X TEA buffer

c. Vortex, let incubate at room temp for 10 minutes

d. During incubation, prepare 7 identical tubes with 800 µL dye solution and 50 µL of 1X TEA

e. After 10 minute incubation is completed, add 7.5 µL of 100 mM ATP to reaction, vortex, start timer (final 2 mM)

f. At 1 min 55 sec (5 seconds before timepoint), remove 50 µL of reaction, at 2 min add it to dye solution tube, vortex, let react for 1 min

g. At 3 minutes, add 100 µL of 34% citric acid to tube, vortex, pipette into cuvette

h. Take 3 readings at 660 nm at 3 min 25 sec, 3 min 30 sec, 3 min 35 sec

i. Repeat steps f, g, and h at 4, 6, 8, 10, 12, and 14 minutes
[[Category:Protocols]]