Operating Manual
19
1300-87076-1A Rev. 1
4. Open the detection arm and turn the pathlength selector to the appropriate position. For example, the BSA
concentration range of 0.5 mm is from 0.06 to 45 mg/mL, and 0.05 mm is from 30 to 600 mg/mL. All the
method ranges are shown on the Spex NanoSNAP screen.
5. Add appropriate solution of at least 1 µL and tap on the
Blank
icon to establish blank data.
6. Tap on the
Name
bar to insert the sample name (optional). The naming system is auto-numbering.
7. Wipe away the blank solution off the sample window and the cover window with a lint-free wipe.
8. Add your sample of at least 1 µL and tap on the
Measure
icon for sample measurement.
9. Clean the sample window and cover window on the detection arm with a lint-free wipe after the
experiment. Use deionized water, ethanol or isopropanol if needed.
10. The
Baseline correction
(340 nm) function is optional and can be turned on/off any time.
11. The default of the
Auto Run
function is off. If
Auto Run
is turned on, sample measurement will be
performed automatically after closing the detection arm.
Note:
1. In Protein A280, BSA, lgG, Lysosome, 1 A = 1 mg/mL, and customized protein factor applications are offered
in this protocol. If you need to test other samples, please use
Factor Method
to customize your protocol
setting in
More Assays
.
2. Blank is not allowed to be used for sample naming.
3. It is suggested to clean the sample window and cover window when exchanging different concentrations of
samples.
5.3 Calculation
In the protein A280 protocol, a modified Beer-Lambert equation is used to calculate concentrations with
absorbance and factor as follows, with or without baseline correction.
Without baseline correction
: c = A280 x ԑ / b
With baseline correction
: c = (A280 - A
Baseline
) x ԑ / b
c
the purified protein concentration in mg/mL
A280
the absorbance at 280 nm
A
Baseline
absorbance at baseline wavelength
ԑ
the extinction coefficient factor/purified protein factor in g x cm/L
b
the pathlength in cm
The extinction coefficient factors used in the calculation of purified protein are shown in Table 11.
Table 11. Extinction coefficient factors of purified proteins.
Type
Extinction Coefficient Factors (g x cm/L)
Extinction Coefficient Factors (L/g x cm)
BSA
1.50
0.667
lgG
0.72
1.37
Lysosome
0.38
0.264
1 A = 1 mg/mL
1
1
