3
TECHNICAL NOTE
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The functionality of the HSAX complex was additionally
tested in the same experiment by adding ATP as a
ligand (Figure 2). The actin, Arp4 and Arp8 subunits
of HSAX are known to bind to ATP.
3,4
The presence of
ATP induced marked changes in the unfolding profile
of HSAX. The previously observed single unfolding
transition changed into two transitions with two clearly
distinguishable, higher inflection temperatures, likely
representing the different stabilizing effects of ATP on
actin and the Arp proteins.
Arp4
Act
Arp8
HSA
N
Strep-tag
30 40 50 60 70 80 90 100
0.86
0.88
0.90
0.92
0.94
0.96
0.98
Ratio
350
nm
/
330
nm
Temperature (°C)
Fraction G7
Fraction F3
Reference HSAX
60 80 100
0
50
100
150
200
250
300
350
F3 F5 F7 F8 F9 F10 G2 G5 G7 G11
elution volume (ml)
mAU
F3
G7
kDa
20
70
50
30
40
25
100
150
200
120
15
10
Marker
pooled
Strep
Elution
FT-Strep
affinity
A B C
D
Reference 0.880 0.095 57.4 N/A
F3 0.879 0.097 57.0 89.5
G7 0.928 0.033
45.6
57.8
36.2
Initial
ratio
T
i
(°C)
Profile
similarity (%)
Ratio Sample
Figure 1: HSAX quality verification during purification using Tycho NT.6. A ) Schematic representation of the HSAX complex. Act = actin, Arp = actin
related protein, HSA = helicase–SANT–associated. B) Elution profile of HSAX a er size-exclusion chromatography with fractions F3 and G7 indicated.
C) SDS-PAGE of fractions from size-exclusion chromatography. D) Unfolding profiles of fractions G7 and F3 compared to a reference sample of purified
HSAX. Measurement results are summarized in the table. T
i
= inflection temperature, the temperature at which an unfolding transition occurs and is
detected by the system.
