TITLE: ATPase activity assay
AUTHORS: Anika Wiegard, Christin Köbler, Katsuaki Oyama, Anja K. Dörrich, Chihiro Azai, Kazuki Terauchi, Annegret Wilde, Ilka Maria Axmann

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol can be used to analyse ATPase activity of KaiC proteins. Produced ADP is separated chromatographically using HPLC and monitored via its absorption at 260 nm.</div></div>

[STEPS]
?. [Preparation of running buffer]
fill a 1l bottle with 963.4 ml MilliQadd 5.8 ml phosphoryc acid (17.2 M stock Nacalai tesque, CAS 121-44-8) [for pH adjustment] --> final concentration = 100 mMadd 20.8 ml trimethylamine (7.15M stock, Nacalai tesque, CAS 7664-38-2) [counter-ion]  --> final concentration = 150 mMadd 10 ml acetonitrile (99.8 % stock, Sigma Aldrich, CAS 75-05-08) [mobile phase] --> final concentration = 1%degas shortly after mixing all samples (0.5-1.5 min) (Note: longer degassing will lead to evaporation of acetonitrile)
?. [Preparation of MiliQ, MeOH (and ETOH)]
fill a 1l bottle with MiliQ and degas for 10 minfill a 1l bottle with 80 % MeOH and degas shortlyoptional: fill a 1l bottle with 20 % EtOH and degas for 10 min
?. [Preparation of the HPLC instrument]
provide MilliQ for piston back flushingconnect pump A, pump B and autosampler to MilliQpurge autosampleropen pump valves, purge and close valves againclean all valves with at least 15 ml MilliQ (flowrate: 0.2-0.8 ml/min)Note: do not exceed the maximal pressure, the column can withstand. If pressure is too high, reduce the flowrate
?. [Equilibration of the column]
connect a Shim-Pack-VP-ODS column (SHIMADZU) (flow rate 0.4 ml/min)wash column with at least 36 ml MiliQ to remove EtOH or MeOH (0.2-0.4 ml/min)connect pump A to running buffer (leave pump and autosampler in MilliQ)open pump valves, purge and close valves againequilibrate column with at least 36 ml running buffer (0.2-0.8 ml/min)Note: do not exceed the maximal pressure, the column can withstand. If pressure is too high, reduce the flowrate
?. [Separation of the sample]
mix 0.2 mg/ml KaiC with 1 mM ATP and 5 mM MgCl2 in reaction bufferoptional: add 0.04 mg/ml KaiA and/or 0.04 mg/ml KaiBNote: total volume can vary between 50-200 µl dependent on your incubation time and number of samplestransfer mixture to reaction vial (make sure to eliminate all air bubbles)place reaction vial in autosampler of your HPLC instrumentseparate the sample using the above mentioned standard parametersfor calculation you will have to substract the auto hydrolis of ATP (see step 9). For this purpose, separate samples containing only 1 mM ATP and 5 mM MgCl2 (but no protein) in reaction buffer. You can do this in paralell or use buffer values from an independent separation if you use the same column, buffer and ATP solution.To follow ADP production over time, program an autosampling shedule (e.g. 3 samples and 3 buffer controls measured every 3 hours as shown in the table below)run a final measurement without injecting a sample and water as running buffer (standard parameters, but 100 % B and 0 µl injection volume)Note: total volume can vary between 50-200 µl dependent on your incubation time and number of samplestransfer mixture to reaction vial (make sure to eliminate all air bubbles)place reaction vial in autosampler of your HPLC instrumentseparate the sample using the above mentioned standard parametersfor calculation you will have to substract the auto hydrolis of ATP (see step 9). For this purpose, separate samples containing only 1 mM ATP and 5 mM MgCl2 (but no protein) in reaction buffer. You can do this in paralell or use buffer values from an independent separation if you use the same column, buffer and ATP solution.To follow ADP production over time, program an autosampling shedule (e.g. 3 samples and 3 buffer controls measured every 3 hours as shown in the table below)run a final measurement without injecting a sample and water as running buffer (standard parameters, but 100 % B and 0 µl injection volume)  ABCDE1Time after starting first measurement [h]
Sample number
Sample name
Incubation time of the sample [h]
action
20
1
buffer-1
0
Insert sample vial, start shedule
30.5
2
buffer-2
0
Insert sample vial shortly before this timepoint
41
3
buffer-30
Insert sample vial shortly before this timepoint
51.5
4
KaiC-1
0
Insert sample vial shortly before this timepoint
62
5
KaiC-2
0
Insert sample vial shortly before this timepoint
72.5
6
KaiC-3
0
Insert sample vial shortly before this timepoint
83
1
buffer-1
3
&nbsp;
93.5
2
buffer-2
3
&nbsp;
104
3
buffer-33
&nbsp;
114.5
4
KaiC-1
3
&nbsp;
125
5
KaiC-2
3
&nbsp;
135.5
6
KaiC-3
3
&nbsp;
146
1
buffer-1
6
&nbsp;
156.5
2
buffer-2
6
&nbsp;
167
3
buffer-36
&nbsp;
177.5
4
KaiC-1
6
&nbsp;
188
5
KaiC-2
6
&nbsp;
198.5
6
KaiC-3
6
&nbsp;
209
1
buffer-1
9
&nbsp;
219.5
2
buffer-2
9
&nbsp;
2210
3
buffer-39
&nbsp;
2310.5
4
KaiC-1
9
&nbsp;
2411
5
KaiC-2
9
&nbsp;
2511.5
6
KaiC-3
9
&nbsp;
2612
1
buffer-1
12
&nbsp;
2712.5
2
buffer-2
12
&nbsp;
2813
3
buffer-312
&nbsp;
2913.5
4
KaiC-1
12
&nbsp;
3014
5
KaiC-2
12
&nbsp;
3114.5
6
KaiC-3
12
&nbsp;
3215
1
buffer-1
15
&nbsp;
3315.5
2
buffer-2
15
&nbsp;
3416
3
buffer-315
&nbsp;
3516.5
4
KaiC-1
15
&nbsp;
3617
5
KaiC-2
15
&nbsp;
3717.5
6
KaiC-3
15
&nbsp;
3818
1
buffer-1
18
&nbsp;
3918.5
2
buffer-2
18
&nbsp;
4019
3
buffer-318
&nbsp;
4119.5
4
KaiC-1
18
&nbsp;
4220
5
KaiC-2
18
&nbsp;
4320.5
6
KaiC-3
18
&nbsp;
4421
1
buffer-1
21
&nbsp;
4521.5
2
buffer-2
21
&nbsp;
4622
3
buffer-321
&nbsp;
4722.5
4
KaiC-1
21
&nbsp;
4823
5
KaiC-2
21
&nbsp;
4923.5
6
KaiC-3
21
&nbsp;
5024
1
buffer-1
24
&nbsp;
5124.5
2
buffer-2
24
&nbsp;
5225
3
buffer-324
&nbsp;
5325.5
4
KaiC-1
24
&nbsp;
5426
5
KaiC-2
24
&nbsp;
5526.5
6
KaiC-3
24
&nbsp;
5627
-
MilliQ, waste
MilliQ, waste
&nbsp;
ABCDE1Time after starting first measurement [h]
Sample number
Sample name
Incubation time of the sample [h]
action
20
1
buffer-1
0
Insert sample vial, start shedule
30.5
2
buffer-2
0
Insert sample vial shortly before this timepoint
41
3
buffer-30
Insert sample vial shortly before this timepoint
51.5
4
KaiC-1
0
Insert sample vial shortly before this timepoint
62
5
KaiC-2
0
Insert sample vial shortly before this timepoint
72.5
6
KaiC-3
0
Insert sample vial shortly before this timepoint
83
1
buffer-1
3
&nbsp;
93.5
2
buffer-2
3
&nbsp;
104
3
buffer-33
&nbsp;
114.5
4
KaiC-1
3
&nbsp;
125
5
KaiC-2
3
&nbsp;
135.5
6
KaiC-3
3
&nbsp;
146
1
buffer-1
6
&nbsp;
156.5
2
buffer-2
6
&nbsp;
167
3
buffer-36
&nbsp;
177.5
4
KaiC-1
6
&nbsp;
188
5
KaiC-2
6
&nbsp;
198.5
6
KaiC-3
6
&nbsp;
209
1
buffer-1
9
&nbsp;
219.5
2
buffer-2
9
&nbsp;
2210
3
buffer-39
&nbsp;
2310.5
4
KaiC-1
9
&nbsp;
2411
5
KaiC-2
9
&nbsp;
2511.5
6
KaiC-3
9
&nbsp;
2612
1
buffer-1
12
&nbsp;
2712.5
2
buffer-2
12
&nbsp;
2813
3
buffer-312
&nbsp;
2913.5
4
KaiC-1
12
&nbsp;
3014
5
KaiC-2
12
&nbsp;
3114.5
6
KaiC-3
12
&nbsp;
3215
1
buffer-1
15
&nbsp;
3315.5
2
buffer-2
15
&nbsp;
3416
3
buffer-315
&nbsp;
3516.5
4
KaiC-1
15
&nbsp;
3617
5
KaiC-2
15
&nbsp;
3717.5
6
KaiC-3
15
&nbsp;
3818
1
buffer-1
18
&nbsp;
3918.5
2
buffer-2
18
&nbsp;
4019
3
buffer-318
&nbsp;
4119.5
4
KaiC-1
18
&nbsp;
4220
5
KaiC-2
18
&nbsp;
4320.5
6
KaiC-3
18
&nbsp;
4421
1
buffer-1
21
&nbsp;
4521.5
2
buffer-2
21
&nbsp;
4622
3
buffer-321
&nbsp;
4722.5
4
KaiC-1
21
&nbsp;
4823
5
KaiC-2
21
&nbsp;
4923.5
6
KaiC-3
21
&nbsp;
5024
1
buffer-1
24
&nbsp;
5124.5
2
buffer-2
24
&nbsp;
5225
3
buffer-324
&nbsp;
5325.5
4
KaiC-1
24
&nbsp;
5426
5
KaiC-2
24
&nbsp;
5526.5
6
KaiC-3
24
&nbsp;
5627
-
MilliQ, waste
MilliQ, waste
&nbsp;
?. [Separation of ADP standards (necessary for calibration curve)]
prepare standard solutions with defined amounts of ADPtransfer to sample vialsplace in autosamplerperform chromatography as described before
?. [Cleaning and storage]
connect pump A to waterpurge autosampleropen pump valves, purge and close pump valves againwash column with at least 36 ml MiliQ (0.4-0.8 ml/min)connect pump B and autosampler to 80 % MeOHpurge autosampleropen pump valves, purge and close pump valves againclean column with at least 36 ml 80 % MeOH (0.1-0.4 ml/min)detach columnoptional: if the capillaries you use, cannot withstand 80 % MeOH for a long time, or if you want to store your column in EtOH, perform the following steps to exchange it to EtOH:wash the column with at least 36 ml MiliQ (0.4-0.8 ml/min)connect pump B 20 % EtOHopen pump valves, purge and close pump valves againwash the column with at least 36 ml 20 % MeOH (0.1-0.4 ml/min)
?. [Data evaluation]
for each timepoint: determine the area of the ADP peak at 260 nm (width 60 sec, slope: 200 nU/min)Note: retention time of ADP is ~14.5 min after separation at 30 °C , but varies with temperature. plot a calibration curve based on your standard measurements (step 7)samples: for each timepoint: substract ADP peak measured in the buffer sample from the ADP peak measured in the protein sample (ADP[protein]-ADP[buffer]=ADP[normalized])for each timepoint: calculate ADP molarity corresponding to ADP[normalized] from the calibration curve (e.g. (ADP[normalized]-10162)/4136.8=µM ADP)plot produced ADP over time, add a linear trend line and note it’s slope (e.g. 1.32)divide slope by the molarity of your protein in the sample to obtain produced ADP per KaiC monomer (e.g. 1.32/3.45µM)multiply by 24 hours to obtain produced ADP per KaiC monomer and dayNote: retention time of ADP is ~14.5 min after separation at 30 °C , but varies with temperature.plot a calibration curve based on your standard measurements (step 7)samples: for each timepoint: substract ADP peak measured in the buffer sample from the ADP peak measured in the protein sample (ADP[protein]-ADP[buffer]=ADP[normalized])for each timepoint: calculate ADP molarity corresponding to ADP[normalized] from the calibration curve (e.g. (ADP[normalized]-10162)/4136.8=µM ADP)plot produced ADP over time, add a linear trend line and note it’s slope (e.g. 1.32)divide slope by the molarity of your protein in the sample to obtain produced ADP per KaiC monomer (e.g. 1.32/3.45µM)multiply by 24 hours to obtain produced ADP per KaiC monomer and day
?. [buffer validation]
to test whether buffer still contains acetonitrile as mobile phase and to control quality of ATP mix 1 mM ADP, 1 mM ATP and 5 mM MgCl2in reaction buffer (20 mM Tris/HCl (pH 8), 150 mM NaCl)       component 
   volume [µl] 
     Reaction buffer 
   97  
     0.1 M ATP 
   1 
     0.1 M ADP 
   1 
     0.5 M MgCl2 
   1 
   separate ADP and ATP using the following standard parameters  AB1  component    volume [µl]  2  Reaction buffer    97   3  0.1 M ATP    1  4  0.1 M ADP    1  5  0.5 M MgCl2    1  standard parameters:  AB1  parameter    Set to  2  pump    100 % A (running
  buffer)  3  flow rate    0.4 ml/min  4  run time    30 min  5  injection volume
  (autosampler)    2 µl  6  depth of the
  injection needle    52 nm (depends on
  your vials and instrument)  7  autosampler
  temperature    30 °C, if not stated
  otherwise  8  column oven
  temperature    30 °C, if not stated
  otherwise  9  PDA detector
  wavelength    190-800 nm (you will
  analyse absorption at 260 nm)  10  PDA detector
  sampling time    Every 0.64 sec
AB1  component    volume [µl]  2  Reaction buffer    97   3  0.1 M ATP    1  4  0.1 M ADP    1  5  0.5 M MgCl2    1
AB1  parameter    Set to  2  pump    100 % A (running
  buffer)  3  flow rate    0.4 ml/min  4  run time    30 min  5  injection volume
  (autosampler)    2 µl  6  depth of the
  injection needle    52 nm (depends on
  your vials and instrument)  7  autosampler
  temperature    30 °C, if not stated
  otherwise  8  column oven
  temperature    30 °C, if not stated
  otherwise  9  PDA detector
  wavelength    190-800 nm (you will
  analyse absorption at 260 nm)  10  PDA detector
  sampling time    Every 0.64 sec