Quantitation of micro-volume protein and DNA samples is important for life science laboratories. The purpose of this study is to analyze labeled and non-labeled proteins using the Shimadzu BioSpec-nano UV-Vis spectrophotometer. Please visit us at www.ssi.shimadzu.com and on Twitter @shimadzussi

1. Rapid Quantitation of
Micro-Volume Protein Samples
John Kinyanjui, PhD, Jeff Head, MS,
Andrew D. Shaff and C. Mark Talbott, PhD
Molecular Spectroscopy Group
Shimadzu Scientific Instruments
Columbia, MD
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2. Introduction
Quantitation of micro-volume protein and DNA samples
is important for life science laboratories. The purpose of
this study is to analyze labeled and non-labeled
proteins using the
Shimadzu BioSpec-nano UV-Vis spectrophotometer.
The measurement technique used is especially suited
for low sample volumes of 1 to 4 µL using adjustable
pathlengths of 0.2 and 0.7mm.
This study focused on a working linear range of 0.5 –
2.1 mg/mL for the non-labeled protein samples and 0.1
- 2.1 mg/mL for the labeled protein samples.
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3. Experimental Conditions
Two types of protein standards were used:
1. Non-Labeled Goat Anti-Rabbit immunoglobulin (IgG)
fragment (A-10533, Invitrogen)
• The molar absorptivity of the protein, ε, = 140,000 M-1cm- 1 and
has a molecular weight of 100,000 g/mol.
2. Labeled Goat Anti-Rabbit immunoglobulin (IgG)
fragment with Alexa Fluor® 647 dye (A-21246, Invitrogen)
• The molar absorptivity of the dye, ε, = 239,000 M-1cm- 1 and
has a molecular weight of 1200 g/mol.
Protein was diluted using SAFC Biosciences Dulbecco’s
Phosphate Buffered Saline solution (DPBS Modified – pH
7.0-7.4; no calcium or magnesium).
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4. Measurement
Absorbance measurements were performed
using a Shimadzu BioSpec-nano
micro-volume UV-Vis spectrophotometer.
• Measurements were carried out at a pathlength
of 0.7 mm.
• However, measurements can be carried out at a
pathlength of 0.2 mm and 5 mm.
• Rapid measurement of 3 seconds (~8 sec cycle time
sample-to-sample) with built-in auto-wiper.
• Both labeled and non-labeled proteins and nucleic
acids can be quantified with formulas built in to the
BioSpec-nano measurement software.
Figure 1. Shimadzu BioSpec-nano
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5. Spectra of Labeled Protein Goat
IgG
Reported values for
stock solution:
Figure 2. Experimental
Results
• Protein concentration is
2 mg/ml.
• Label - protein ratio is
3 moles label to 1 mole of
protein.
Alexa Fluor® Label (647 nm)
Result output
Protein (~280 nm)
Reported value also denoted
as theoretical value.
Table 1. Experimental
Results
Theoretical
(mg/ml) Experimental
(mg/mL)
%
RSD
Exp:
Ratio
label/protein
(moles)
0.1
0.1
19.4
2.5
0.5
0.5
4.4
3.0
1
1.1
3.1
3.2
1.5
1.6
2.1
3.3
2
2.1
4.4
3.3
%
RSD
20.1
3.4
2.7
1.9
3.0
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6. Results – Labeled Protein Goat IgG
Figure 3. Linear Calibration Curve for Labeled
Goat IgG
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7. Spectra of Non-Labeled Protein Goat
IgG
Reported values for stock
solution:
• Protein concentration
is 2 mg/ml.
Reported value also
denoted as a theoretical
value.
Result output
Table 2. Experimental Results
Theoretical
(mg/ml)
0.5
1
1.5
2
Experimental
(mg/mL)
0.5
1.0
1.6
2.0
%
RSD
3.7
4.9
2.3
5.7
Figure 4. Experimental
Results
Protein (~280 nm)
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8. Results – Non-Labeled Protein Goat
IgG
Figure 5. Linear Calibration Curve for Non-Labeled
Goat IgG
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9. Conclusion
The Shimadzu BioSpec-nano micro volume spectrophotometer can be used to
rapidly and accurately quantify protein concentrations for both labeled and
non-labeled proteins.
The mole concentration ratio of the Alexa Fluor® Label to the Goat IgG is in
good agreement to the manufacturer-supplied value of 3.
The measurement accuracy is similar for both the labeled and non-labeled
Goat IgG.
Although values as low as 100ng/mL could be measured for the labeled Goat
IgG, a higher RSD is reported on the lower end of the linear measurement
range.
The lower range values obtained for the linear calibration curves for both protein
types are well within lower values of the reported dynamic range for the
BioSpec-nano of 0.45 – 31.8 mg/ml.
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research, products or services, please visit our support
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