Low-volume protein measurements (280nm):
Validating the LVis Plate over many concentrations
Sarfaraj Topia, Alison Turner- UCB Pharma, Bath Road, Slough
Different instrumentation was used to measure UV protein
absorbance at 280 nm
Low and high range protein concentrations were successfully
measured in 2 µl with the LVis plate on the FLUOstar Omega
microplate reader
Introduction
Measurement at the working concentration of an experiment in a high
throughput manner is useful especially when dilution of the protein
may affect the solubility characteristics. This would also be
advantageous where accurate protein concentration is required on
numerous samples (for example, column elution fractions), where
dilution would be time consuming and potentially introduce pipetting
errors. This coupled with the ability of being able to analyse the
samples at low volume would also offer an added advantage
when quantities of sample are limited.
The LVis Plate is a proprietary absorbance microplate from BMG
LABTECH and is perfectly suited for low volume protein quantitation.
It consists of sixteen microdrop well sites for 2 µL samples and adheres
to the 96-well microplate format definition. The microdrop well sites
are also easily accessible to wipe clean for further measurements.
The aim was to demonstrate that the LVis Plate was able to measure
protein concentration at two different concentration ranges (0.2 to
18 mg/mL) reliably as compared to alternative methods, a UV/Vis
spectrophotometer (manual cuvette sampling), and an alternative
high throughput technique (NanoDrop).
Application Note 242, Rev. 07/2013
Serial dilutions of the protein stock solution were made ranging
from 0.2 mg/mL to 1.0 mg/mL in buffer (detailed above). The
absorbance at 280 nm was measured using the LVis plate (BMG
LABTECH) and compared with results obtained from alternative
spectrophotometers, a UV/Vis Spectrophotometer and the
NanoDrop. Measurements were made in triplicate for each method.
Aliquots of 2 µL were used for the LVis Plate and the NanoDrop,
whereas 100 µL was used for the UV/Vis spectrophotometer.
In each case the samples were blanked against the buffer
(detailed above).
The mean and the standard deviation of the absorbance readings
at 280 nm at each concentration for each method was derived and
then plotted against nominal concentration (Figures 2 - 5).
Experiment 2
To compare reproducibility at a higher concentration range (up to
18 mg/mL).
This experiment only compared the LVis Plate and NanoDrop since the
linearity of the UV/Vis spectrophotometer is known to be unreliable
at the higher concentration range and hence would not provide a fair
test. Dilutions of the protein stock were made from 18 mg/mL to
1 mg/mL in buffer (detailed above). Triplicate measurements were
made and the data plotted as in Experiment 1 (Figure 6).
A
B
The LVis Plate was used with the FLUOstar OMEGA microplate reader
(BMG LABTECH), which has an ultra-fast UV/Vis spectrometer. This
technology captures an entire spectrum (220 - 1000 nm) in
<1 sec/well. The LVis Plate is also compatible with the SPECTROstar
Nano, PHERAstar FS, and CLARIOstar microplate readers from
BMG LABTECH.
Materials and Methods
Fig. 1: (A) BMG LABTECH’s multidetection microplate reader with UV/Vis
Absorbance spectrometer and (B) BMG LABTECH’s LVis Plate.
Sample Protein Stock Solution 30 mg/mL in 20 mM sodium acetate, 100 mM sodium chloride, pH 5.0
Working buffer : 50 mM sodium acetate, 100 mM sodium chloride, pH 5.0 [filtered (0.22µm)]
LVis Plate ( BMG LABTECH)
Cary Bio 50, UV/Vis Spectrophotometer
NanoDrop, Thermo Scientific, UK
Experiment 1
At concentrations ranging from 0.2 mg/mL and 1.0 mg/mL, the mean
absorbance at 280 nm (OD) was plotted against the concentration and
the linear regression fit was calculated.
Experiment 1
To compare the reproducibility at a low concentration range (up to
1 mg/mL). The sample protein is a full length monoclonal antibody.
A high R squared value (R2) in excess of 0.99 was obtained for the
UV/Vis Spectrophotometer (Figure 2), LVis Plate (Figure 3) and the
NanoDrop (Figure 4) .
Results and Discussion
An overlay of the data obtained from each measurement technique
(Figure 5) showed that there was good correlation between the
three methods.
UV/Vis Spectrophotometer
R2 = 0.9993
1.4
The R squared values (R2) were in excess of 0.99 for both the LVis
Plate and NanoDrop and good correlation was observed between
the two methods as illustrated by the overlaid results (Figure 6).
1.2
OD at 280nm
Experiment 2
This experiment was performed to check the reliability of the LVis
Plate to measure the absorbance at higher concentrations (up to
18 mg/mL) compared with the Nano Drop only. The mean absorbance
values at different concentrations were plotted against the backcalculated concentration to show linearity.
1
0.8
0.6
0.4
LVis Plate Validation
0
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
Protein (mg/mL)
Fig. 2: Results obtained through Cary 50 Bio Spectrophotometer
LVis Plate
R2 = 0.9991
1.4
High Protein Concentration
Experiment 2
25
20
15
NanoDrop
R2 = 0.9994
10
LVis Plate
R2 = 0.9996
5
0
0
1.2
OD at 280nm
Back-Calculated Protein (mg/mL)
0.2
5
10
15
20
Protein (mg/mL)
1
Fig. 6: Overlapped profiles of results obtained from LVis Plate and NanoDrop
0.8
0.6
0.4
0.2
0
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
Protein (mg/mL)
Conclusion
All three instruments for the measurement of concentrations up to
1 mg/mL showed equivalent results.
Fig. 3: Result obtained from LVis Plate from BMG LABTECH, UK
At concentrations up to 18 mg/mL, the LVis Plate and the Nano Drop
(an alternative high throughput method) showed comparable results.
NanoDrop
Overall, the LVis Plate proved to be a reliable, low volume high
throughput method for measurement of protein concentrations
from 0.2 mg/mL to 18 mg/mL. This was validated against existing
alternative spectrophotometric techniques.
R2 = 0.9985
1.4
OD at 280nm
1.2
1
0.8
0.6
0.4
0.2
0
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
Protein (mg/mL)
Fig. 4: Results obtained from NanoDrop from Thermo Scientific, UK
LVis Plate Validation
Low Protein Concentration
Experiment 1
OD at 280nm
1.5
1
UV/Vis Spec
NanoDrop
0.5
LVis Plate
0
0
0.2
0.4
0.6
0.8
1
Protein (mg/mL)
Fig. 5: Overlapped profiles of all three measurement techniques
1.2
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