Rapid Quantitation of
Micro-Volume Protein Samples
John Kinyanjui, PhD, Jeff Head, MS,
Andrew D. Shaff and C. Mark Talbott...
Introduction
Quantitation of micro-volume protein and DNA samples
is important for life science laboratories. The purpose ...
Experimental Conditions
Two types of protein standards were used:
1. Non-Labeled Goat Anti-Rabbit immunoglobulin (IgG)
fra...
Measurement
Absorbance measurements were performed
using a Shimadzu BioSpec-nano
micro-volume UV-Vis spectrophotometer.
• ...
Spectra of Labeled Protein Goat
IgG
Reported values for
stock solution:

Figure 2. Experimental
Results

•  Protein concen...
Results – Labeled Protein Goat IgG

Figure 3. Linear Calibration Curve for Labeled
Goat IgG

6 / 10
Spectra of Non-Labeled Protein Goat
IgG
Reported values for stock
solution:
•  Protein concentration
is 2 mg/ml.

Reported...
Results – Non-Labeled Protein Goat
IgG

Figure 5. Linear Calibration Curve for Non-Labeled
Goat IgG

8 / 10
Conclusion
The Shimadzu BioSpec-nano micro volume spectrophotometer can be used to
rapidly and accurately quantify protein...
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any questions or require additional information about our
researc...
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Rapid Quantitation of Micro-Volume Protein Samples

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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.

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Rapid Quantitation of Micro-Volume Protein Samples

  1. 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 1 / 10
  2. 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. 2 / 10
  3. 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). 3 / 10
  4. 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 4 / 10
  5. 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 5 / 10
  6. 6. Results – Labeled Protein Goat IgG Figure 3. Linear Calibration Curve for Labeled Goat IgG 6 / 10
  7. 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) 7 / 10
  8. 8. Results – Non-Labeled Protein Goat IgG Figure 5. Linear Calibration Curve for Non-Labeled Goat IgG 8 / 10
  9. 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. 9 / 10
  10. 10. Thank you for viewing this presentation. Should you have any questions or require additional information about our research, products or services, please visit our support page: www.ssi.shimadzu.com/support/ Follow us on Twitter @shimadzussi 10 / 10

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