P. Fajer
page 1 of 7
Solutions for ATPase Assay.
Solutions
Please use only the special glassware marked “For malachite green only”. After use rinse everything you used with
1M KOH/50% ETOH then dH20.
0.45% Malachite Green:
Use malachite green hydrochloride (Sigma #M-9636). 0.45g in 1000ml dH20.
Store at room temp.
34mM Ammonium Molybdate in 4N HCl: Use Molybdic acid, ammonium salt (Sigma #M-0878). Prepare 4N HCl by
diluting conc. HCl (12N) by 3. Do this in the fume hood, wear gloves, add acid to
H20. Add 42g ammonium molybdate to 1000ml dH20, stir, add 37.8g ammonium
molybdate. Mix well, store at room temperature.
Sterox detergent:
Use Perkin Elmer #C051-0150, store at room temp.
MG-AM-ST:
Add 300ml 0.045% malachite green solution to 100ml 34mM ammonium
molybdate in 4N HCl (or any 3:1 mix). Stir for 20 min at room temp. Filter through
Whatman #5 filter paper. Add 8ml sterox to 400ml MG-AM (1:50 ratio). Store at
4C in a dark bottle.
34% Sodium Citrate:
Use citric acid, tri-sodium salt (Sigma #C-0909). 68 g in 200ml final volume. Store
at room temp.
1M KOH/50% ETOH:
First prepare 1M KOH. For 100ml final volume use 5.6g K OH. Prepare a 50:50
mixture of 1M KOH : ethanol, e.g.100ml 1M KOH plus 100ml ethanol.
106741197
2/16/2016 10:45:00 PM2/16/2016 10:45:00 PM
P. Fajer
page 2 of 7
ATPase Assay
For the changes necessary for the 200l assay see 200l data sheets.
Ref:
Lanzetta, et. al, Anal. Biochem. 100, 95 (1979).
Reagents:
FOR RECIPES SEE ATPASE.SOL (Disk = Wet Lab Methods).
1)
distilled H20.
2)
Mg-AM-ST (in Repipette II bottle, stored in the cold room).
3)
0.4mM phosphate standard (prepared from.65mM aliquots stored in freezer door).
4)
100mM and 5.4mM ATP (the 100mM ATP is in aliquots in the freezer door, the 5.4mM ATP is
made form the 100mM ATP.
5)
A23187 (this is for SR only and is stored in the freezer as 1 mg/ml in DMF).
6)
ATPase media x 2 (stored in the cold room on the ATPase shelf).
Type of ATPase
ATPase Media
+Ca
SR
high salt myosin and
and Ca
K/EDTA and K/Ca
high salt myofibril
high salt S1
physiological myosin and
R2505 and C1505
physiological myofibril
Materials needed:
1)
magnetic fleas.
2)
25C water bath.
3)
One pipetteman pipette to deliver 500l aliquots (P200 or P100) and one to deliver 200l
aliquots (P1000 or P2000).
4)
stopwatch.
Assay:
1)
Thaw ATP and Pi standard. Make 5.4mM ATP by adding 50l of 100mM ATP to 872l of dH20 in
an eppendorf tube.
2)
Prepare ASSAY TUBE: place 1.6ml (using Repipette II bottle) of Mg-AM-ST into test tubes; use
one for each time point plus two for the blanks, and at least two for the Pi standards. (See #5.)
106741197
2/16/2016 10:45:00 PM2/16/2016 10:45:00 PM
P. Fajer
page 3 of 7
INCUBATION TUBES
(ml media
ml
ml
ml
mg
x2)
H2 0
other1
sample
sample
0.50
0.45  (x + y)
y
x
xc*
Total volume = 0.95ml
Make duplicates at least.
1E.G.
A23187 (standard for SR: usually 2l of 1mg/ml A23187 in DMF/.1mg protein)
*c = concentration of protein stock solution in mg/ml.
If possible choose x, c, and assay time t so that A(660) is in the range 0.1-1.
If using t = 0, 1, 2, 4 minutes (recommended), choose xc so that a(660) is ~0.2 in 1 minute.
Since for 50l
I. U. 
A net * 0.4
then x * c 
x*c*t
A net * 0.4
I. U.*t
where Anet = A(t = 1)  A(t = 0).
Use the following table as an approximate guide:
Ca
x*c
TYPE of ATPase (25)
+Ca
I.U.
(mg)
x*c
I.U.
(mg)
SR
.05
.1
.05
2.5
High salt, unlabeled myosin
.05
1.5-2
.10
.25
High salt, SH1-labeled myosin
.10
.2-.4
.05
1-21
High salt, unlabeled myofibrils2
.03
.6
.03
.07
.03
.05-.1
.03
.25
*
.03
(same)
Phys, SH1-labeled myosin2
*
.1
(same)
Phys, unlabeled myofibrils
.4
.02
.2
.4
Phys, SH1-labeled myofibrils
.4
.04
.2
.28
.025
.6
.012
~2.5
High salt, SH1-labeled
Phys unlabeled
myofibrils2
myosin2
………………
.012
MSL-S1, High Salt
.025
.1-.2
1Lower
for MSL
2These
are done using the 200 microliter assay (Don’t add more protein than recommended!).
*Not enough data yet.
4)
Equilibrate at 25C bath for 5.
5)
While first tubes are warming up assay blanks and standards. For blanks use 50l d H20 in assay
tube, vortex, wait 30 sec., add 200l 34% citrate, vortex. For standards most people use 50l 4mM Pi and do
duplicates. One could also use 25l dH20 + 25L.4mM Pi and 75l.4mM Pi to check linearity of the assay. The 75l
standard is necessary if absorbance over 1.0 are obtained during the assay. Be careful to read absorbance of the
106741197
2/16/2016 10:45:00 PM2/16/2016 10:45:00 PM
P. Fajer
page 4 of 7
assay tubes within ~20-30 when working at high absorbance as the green complex readily falls out of solution at
high concentrations.
6)
Assay zero time point: Add 50l unactivated incubation solution to assay tube, then add 50l 5.4mM
ATP vortex; wait 30 sec. Add 200l 34% citrate; vortex.
7)
Activate ATPase Enzyme: Add 50l 0.1M ATP at time = 0 (time it) to INCUBATION TUBE, vortex,
return to water bath and stir gently with a magnetic flea. Change pipette tip.
8)
Assay other time points: Add 50l to assay tube from INCUBATION TUBE at exactly time = t
minutes; vortex. Wait 30 sec. After the addition, add 200ml 34% citrate and vortex.
9)
Read absorbance at 550nm after ~15 min. Zero absorbance by reading blank vs. blank. Leave
blank in reference cuvette and read absorbance for all tubes. If A(660) > 1, either make Pi
standards of the same absorbance or discard the value.
10)
Activity = I.U. = (moles Pi assayed)/(mg protein assayed)/min. 10.1)

A( 660, sample)  A( 660, t  0)
* ( ml st. assayed ) * ( Mmole sample st / ml )
A( 660, s tan dard )
( = 200 for 200 microliter Assay)
/ (mg protein in 1 ml inc.) * (0.5 ml inc. assayed) / t(min)
But since (ml st. assayed) = (ml inc. assayed) and st. is 0.4mM, this reduces to

10.2)
A ( 660), sample)  A ( 660, t  0)
0.4
1
*
*
A ( 660, st.)
mg prot. in inc t(min)
Either
…………………………………………………………………………………….
(Note: If vols. of standard or sample assayed change, or concentration of standard is changed, use
Eq. 10.1 and insert correct values.)
c)
Use ATPLPT. For both B and C, discarding bad data points. (Use mg protein when
the program asks for mg/ml in inc. tube.)
K / EDTA activity of labeled sample
K / EDTA activity of unlabeled control
11)
% SH1 Modified = 1 
12)
With several assumptions the fraction of dead heads, which is taken to be those with both SH1 and
SH2 modified, can be calculated. [Ref: Crowder & Cooke, J. Muscle Research & Cell Motility, 5,
131-146 (1984).]
z 
106741197
1 / c  f k  f c   1  f k 
2/16/2016 10:45:00 PM2/16/2016 10:45:00 PM
P. Fajer
page 5 of 7
where z = fraction dead heads
c = degree of activation of the Ca/K activity as a result of SH-1 modification, taken
to be 10
13)
fk =
K / EDTA activity of labeled sample
K / EDTA activity of unlabeled control
fc =
Ca / K activity of labeled sample
Ca / K activity of unlabeled control
specificity = S =
fSH1 mod ified
1
spins / head
Notes
1)
In all cases, the goal is to measure initial rate. So in discarding bad points be especially careful to discard
low values at long times. Also discard values of A > 1 unless you have a standard P curve in that range.
Also beware of precipitation at Abs. >> 1.
2)
In the wet lab freezer is a small amount of unlabeled myosin in SMB at a know protein
concentration (listed on the storage tube). Use this myosin regularly when doing assays as a standard and
record the activity in the MYO, FRAG & ACTIN PREPS notebook. If you value looks out of line with the rest
then you will want to consider repeating the assay, double checking protein concentrations, etc. This
control is especially important for the high salt myofibril ATPases which have been showing some variability.
Also for these particular assays a control of ground myofibrils in RB/2 + 50% glycerol (also in the wet lab
freezer) should be used as a further standard.
3)
RECORD ALL ACTIVITIES IN THE APPROPRIATE NOTEBOOKS: MYOSIN, FRAGMENTS, &
ACTIN PREPS or FIBER PREPS. Diligence in doing this will help you and others know when there is a
problem with ATPase results before writing
106741197
2/16/2016 10:45:00 PM2/16/2016 10:45:00 PM
P. Fajer
page 6 of 7
Actin Activated ATPase
1.
Use myosin …………… actin in …….
2.
Take 10ml 1mg/ml myosin by diluting an aliquot of M (pipette with Wiretrol) into …….. +.05M KCl. Stir with
Wiretrol.
3.
Make Actomyosin. Use Actin/myosin ratios of 0, 5, 10, 20. To determine amount of actin to add to 2ml of
myosin use:
V=M
[myo]mg/ml x 42.5 x 103(MW actin) x 2ml
A
(actin 1mg/ml) 480.0 x 103(MW myosin)
Where: …. = the ratio of actin to myosin;
…. = the volume of actin to ………;
…. = the volume of myosin (2ml).
Example: Actin ……………………
COULD NOT READ TABLE ACCURATELY
Add buffer, them …. Then ………. Gently by aspirating into a Pasteur pipette.
4.
Save …. ……………………………………………………………………needed plus some extra for
…………………..pipetting). Place in ………………….., seal tightly and store on ice.
5.
Cut dialysis bags and let bags soak in dialysis medium (rb ….60 mM KCl) for a few minutes. Transfer AM
samples into bags with Pasteur pipette, clamp off bag as close as possible to top of liquid. Dialyze against 100
volumes of RB(KCl) overnight.
6.
Remove dialysis bags from buffer and wipe thoroughly. Empty bag into test-tube.
7.
ATPase ……………………………………
a)
Make ……………………………………
b)
…………………….into each assay tube.
c)
…………..sample into incubation tube with magnetic flea.
d)
…………………RB into ………………………….wait 30 sec, add 200l)
Protein Determination
Biuret B
dH20
FDS
Protein
 5 (ml)
(ml)
(ml)
(ml)
Blank
1.0
0.5
BSA-1
1.0
0.39
0.1
0.01
BSA-2
1.0
0.38
0.1
0.02
BSA-3
1.0
0.37
0.1
0.03
Sample 1
1.0
0.40
------
0.10
106741197
2/16/2016 10:45:00 PM2/16/2016 10:45:00 PM
A310
A310
AAVE
P. Fajer
page 7 of 7
Sample 2
1.0
0.40
------
0.10
Sample 3
1.0
0.40
------
0.10
ATPase Assay
Incubation tubes: - Add ATP at time zero. Want 1ml total.
0.5ml K+EDTA (+/- Ca2+) x 2 (in cold room)
(0.45-x) ml dH20
x ml protein (want 0.01-0.05 mg)
0.05 ml 0.1M ATP (add at time zero)
Add magnetic flea, place in 25C water bath for 5min. to equilibrate. Add ATP at time zero.
Assay tubes: Contain 1.6 ml MG/AM/ST at room temp.
Blanks: - To assay tubes add 50l dH20 (wait 30 sec;
add 200l 34% citrate.
Standards: - To assay tubes add 50l 0.4mM PPi (wait 30 sec.)
add 200 l 34% citrate.
Zero Time Point: - To assay tubes add 50l unactivated incubation mixture;
add 50l %*5.4mM ATP (wait 30 sec.)
add 200l 34% citrate.
*To make 5.4mM ATP use 50l 0.1M ATP plus 0.872ml dH20.
After incubation tube has equilibrated: - Add ATP at time zero.
Take 50l aliquots at each time-point.
Wait 30 sec. Then add 200l citrate.
106741197
2/16/2016 10:45:00 PM2/16/2016 10:45:00 PM