Using Infinite® 200 NanoQuant for Absorbance-Based Protein Detection in Small-Volume Samples

Introduction

The availability of new tools has greatly facilitated the quantitative analyses of proteins and nucleic acids. Such tools need only small amounts of sample for reliable, accurate, and reproducible quantification.

Tecan’s Infinite® 200 NanoQuant in tandem with the NanoQuant Plateis one of the devices suited for measuring small-volume samples. It delivers excellent performance for the assessment of labeling efficiency and absorbance-based quantification of nucleic acids.

Tecan’s NanoQuant Plate

The NanoQuant can also be upgraded according to user’s convenience with luminescence and fluorescence reading functions. The NanoQuant detection technology developed by Tecan enables the quantification of biomolecules in small-volume samples.

This article describes the use of the NanoQuant Plate for measuring proteins with regard to important assay parameters such as uniformity, linearity, and reproducibility.

Assay principle

In clinical research and basic science applications, absorbance-based quantification of proteins is very common. Ultraviolet (UV) light is absorbed by proteins at 280 nm. This characteristic can be used as the basis for easy and rapid photometric quantification of protein samples with unknown concentration.

The UV absorbance of protein samples is a function of the amino acid content of the protein analyzed, which is different from that of nucleic acids. Using extinction coefficients, nucleic acids can be directly measured by converting absorbance to concentration.

The fraction of tryptophan, tyrosin and, to a lesser extent, phenylalanine significantly affects the absorbance at 280 nm by proteins, resulting in substantial protein-to-protein variations. A different extinction coefficient may be required by each purified protein.

The Lambert-Beer law is not sufficiently accurate and therefore, cannot be used over a wide concentration range with any type of protein sample, although there is a proportional relation between concentration and absorbance for a given protein dissolved in a given substance.

Therefore, dilutions of standard proteins should be included in protein quantifications with the NanoQuant Plate, so that the concentration of unknown samples can be extrapolated.

Materials and methods

Instrument and plates

  • NanoQuant Plate with 16 individual sample positions with integrated quartz optics (Tecan Austria)
  • Infinite M200 NanoQuant Quad4 monochromators microplate reader (Tecan Austria)

Reagents and additional materials

  • BSA, bovine serum albumin (BSA; 50 mg/ml)
  • 70% ethanol
  • ddH2O
  • laboratory paper towels

Dilution series of standard protein

BSA, used as a standard protein, was serially diluted 1+1 in ddH2O. This is summarized in Table 1.

Table 1. Dilution series of BSA

Dilution

final conc. [mg/ml]

Pipetted

A

50

200 μl undiluted stock

B

25

100 μl A + 100 μl H2O

C

12.5

100 μl B + 100 μl H2O

D

6.25

100 μl C + 100 μl H2O

E

3.13

100 μl D + 100 μl H2O

F

1.56

100 μl E + 100 μl H2O

G

0.78

100 μl F + 100 μl H2O

H

0.39

100 μl G + 100 μl H2O

I

0.20

100 μl H + 100 μl H2O

J

0.10

100 μl I + 100 μl H2O

K

0 (blank)

100 μl H2O

 

Measurement parameters and settings

The NanoQuant Plate was used in standard i-control mode for all measurements. Two replicates were used to perform all measurements; ddH2O was used for blanking. Before taking all the measurements, high-pressure, compressed air and an ultrasonic bath were used to clean the NanoQuant Plate according to the corresponding Quick Guide instructions [1].

A measurement script was set up in i-control V1.5 in accordance with the parameters shown in Table 2, with a reference wavelength at 310 nm and a measurement wavelength at 280 nm for correction.

Table 2. Measurement parameters and instrument settings on Infinite M200 NanoQuant with Tecan i-controlsoftware

Measurements on Infinite M200

Parameter

Plate

Part of the plate

Well

Absorbance

Bandwidth

Flashes

Absorbance

Bandwidth

Flashes

Setting

Tecan 16 Flat Black [NanoQuant Plate]

A1-H2

 

280 nm

5 nm

25

310 nm

5 nm

25

 

Results

The ability of the NanoQuant Plateto measure the protein content of small-volume samples was tested. To assess the measurement performance of the NanoQuant Plate for uniformity, linearity, and reproducibility, the bovine serum albumin (BSA) was used as a standard protein.

Calculations

The following calculations were used to evaluate the results.

All OD280 values, including the blank values, were corrected by the corresponding OD value at 310 nm:

Each protein-containing sample was also corrected for the 310 nm-corrected OD values of the blanks:

Microsoft Excel was used to calculate standard deviations, average results, and coefficients of variation (CV).

Linearity

A line diagram was used to plot blank- and 310 nm-corrected OD280 values; a trend line was also added. Coefficient of determination (R2) and slope were calculated. In Figure 1, good linearity values are seen for BSA dilutions spanning from 50 to 0.1 mg/ml, leading to an R2 value of 0.9976.

Figure 1. Absorbance linearity of standard dilution series (50 – 0.1 mg/ml BSA) at 280 nm after blank- and 310 nm-correction

Additionally, a series of protein samples were prepared and the OD280 values obtained were corrected for blanks and 310 nm absorbance in the same manner as that of the standards used for the dilution series. The measured OD values (y) were inserted into the ascending slope formula of the standard dilutions to determine the effective concentrations (x) (Figure 1).

Note: The factor F for the protein concentration calculation relies on the solvent, standards, and the kind of protein used.

Table 3 summarizes the results and the deviations to the originally pipetted concentrations.

Table 3. Accuracy of protein sample quantification with various BSA concentrations

Sample

OD280_blanked

Effective [c]

[mg/ml]

Theoretical [c]

[mg/ml]

% deviation

1

1,3910

48,98

50

2,04

2

0,8696

30,62

30

2,07

3

0,7452

26,24

25

4,97

4

0,4433

15,61

15

4,08

5

0,2881

10,14

10

1,44

6

0,2453

8,64

8

7,98

7

0,1789

6,30

6

5,03

8

0,1463

5,15

5

3,04

9

0,0891

3,14

3

4,51

10

0,0287

1,01

1

1,20

11

0,0236

0,83

0,8

4,17

12

0,0141

0,50

0,5

0,23

 

The analyzed samples, in a concentration range of 50 to 0.5 mg/ml, show deviations of less than 10% to the theoretical protein concentrations, that is, the known dilutions of the BSA standard. Due to substantial results variation (CV > 10%), samples below 0.5 mg/ml cannot be measured reliably.

Uniformity

A representative protein sample was applied to all 16 sample positions to evaluate the uniformity of protein sample quantification on the NanoQuant Plate. Absorbance was read respectively at 280 nm and 310 nm, and the results were blanked and 310 nm-corrected as illustrated before. Table 4 summarizes the calculated average, standard deviation, and CV.

Table 4. Measurement uniformity over 16 samples on the NQP

The very low results variation of 0,89% (CV) show that the NanoQuant Plate has excellent measurement uniformity for protein samples.

Reproducibility

Another set of experiments was performed to determine the reproducibility of protein quantification measurements on the NanoQuant Plate in which a protein sample with 10 mg/ml was employed in five consecutive measurements, each of which contained four independent sample replicates. Figure 2 shows the plotting of blank- and 310 nm-corrected average OD280 values.

Figure 2. Measurement reproducibility of 5 individual samples on the NanoQuant Plate

Figure 2 shows that there is very little variation between the single measurements which results in a CV of 0.78%. Therefore, the NanoQuant Plate shows very good reproducibility in terms of protein quantification.

Conclusion

This article has shown how Tecan’s NanoQuant Plate serves as an excellent tool to quantify the nucleic acids present in small-volume samples. Additionally, the NanoQuant Plate’s capacity to measure protein samples has now been tested.

The presented results show that the NanoQuant Plate can quantify proteins through their absorbance at 280 nm. They also show that the NanoQuant Plate can quantify protein samples and eliminate the dilution process, and produce very good results in terms of measurement uniformity, linearity, and reproducibility.

The recommended practice for reliable and reproducible analysis is to create a standard curve of a protein with known concentration to accurately quantify the target samples.

To sum up, the NanoQuant Plate has a wide range of applications, from labeling efficiency measurements and quantification of nucleic acids to quantitative analysis of protein samples.

Abbreviations

  • BSA     bovine serum albumin
  • CV        coefficient of variation
  • NQP     NanoQuant Plate
  • OD       optical density
  • stdev    standard deviation

Literature

[1] Quick Guide NanoQuant Plate™, 2008 (Tecan Austria)

About Tecan

Tecan

Tecan is a leading global provider of automated laboratory instruments and solutions. Their systems and components help people working in clinical diagnostics, basic and translational research and drug discovery bring their science to life.

In particular, they develop, produce, market and support automated workflow solutions that empower laboratories to achieve more. Their Cavro branded instrument components are chosen by leading instrumentation suppliers across multiple disciplines.

They work side by side with a range of clients, including diagnostic laboratories, pharmaceutical and biotechnology companies and university research centers. Their expertise extends to developing and manufacturing OEM instruments and components, marketed by their partner companies. Whatever the project – large or small, simple or complex – helping their clients to achieve their goals comes first.

They hold a leading position in all the sectors they work in and have changed the way things are done in research and development labs around the world. In diagnostics, for instance, they have raised the bar when it comes to the reproducibility and throughput of testing.

In under four decades Tecan has grown from a Swiss family business to a brand that is well established on the global stage of life sciences. From pioneering days on a farm to the leading role our business assumes today – empowering research, diagnostics and many applied markets around the world


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Last updated: Jun 20, 2017 at 5:48 AM

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