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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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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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Experimental Conditions

Two types of protein standards were used: Protein was diluted using SAFC Biosciences Dulbecco’s Phosphate Buffered Saline solution (DPBS Modified – pH 7.0-7.4; no calcium or magnesium).

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.

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Measurement

Figure 1. Shimadzu BioSpec-nano

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.

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Spectra of Labeled Protein Goat IgG

Alexa Fluor® Label (647 nm)

Protein (~280 nm) Result output

Theoretical  (mg/ml) Experimental  (mg/mL) %  RSD Exp:  Ratio  label/protein  (moles) %  RSD0.1 0.1 19.4 2.5 20.10.5 0.5 4.4 3.0 3.41 1.1 3.1 3.2 2.71.5 1.6 2.1 3.3 1.92 2.1 4.4 3.3 3.0

Reported values for stock solution:

•  Protein concentration is 2 mg/ml.

•  Label - protein ratio is 3 moles label to 1 mole of protein.

Reported value also denoted as theoretical value.

Table 1. Experimental Results

Figure 2. Experimental Results

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Goat IgG

Results – Labeled Protein Goat IgG

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Spectra of Non-Labeled Protein Goat IgG

Protein (~280 nm) Result output

Theoretical  (mg/ml) Experimental  (mg/mL) %  RSD0.5 0.5 3.71 1.0 4.91.5 1.6 2.32 2.0 5.7

Reported values for stock solution:

•  Protein concentration is 2 mg/ml.

Reported value also denoted as a theoretical value.

Table 2. Experimental Results

Figure 4. Experimental Results

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Results – Non-Labeled Protein Goat IgG

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

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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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