Using Different Absorbance-Based Assays for Protein Quantification

Introduction

It is necessary to perform protein quantification before continuing with protein samples for isolation, electrophoretic or chromatographic analysis, or immunohistochemical techniques. Colorimetric detection and quantification of proteins can be carried out by two different techniques: protein-copper chelation and protein-dye binding.

Tecan’s Infinite M200 instrument

This article shows how Tecan’s Infinite F200 and Infinite M200 instruments enables easy and sensitive quantification of proteins using various absorbance-based assays, as shown in Table 1.

Table 1. Summary of the three protein assays. Shown are the different absorption maxima, the resulting color of the reagent and the linearity range. A standard curve has to be prepared for each assay to calculate unknown protein concentration.

Assay

Abs-max (nm)

Color

Linearity (μg/ml BSA)

BCA

562

purple

25-2000

Lowry

750

blue

1-1500

Bradford

595

blue

1.2-10

Bradford

595

blue

8-80

BCA protein assay

Bicinchoninic acid (BCA) is used in the BCA Protein Assay for colorimetric quantification of total protein in a sample [1]. In this method, Cu2+ is reduced to Cu1+ by protein in an alkaline medium. Cu1+ complexes with BCA and forms a colored water-soluble chelate, which can be determined at its absorption maximum at 562 nm, as shown in Figure 1.

A 2 ml test tube is used to detect the linearity of protein concentrations within a 20-2,000 μg/ml range. The detection range is approximately 125-2,000 μg/ml, if protein quantification is required in smaller volumes.

Figure 1. Absorbance scan (300-900 nm) of BCA with (▬) and without (▬) BSA on Infinite M200 reader.

If protein samples contain one or more detergents, then the BCA Protein Assay may be the best choice.

Modified Lowry protein assay

The original protocol of Oliver Lowry’s method forms the basis of the Modified Lowry Protein Assay, which happens to be a modified, more stable formulated product [2].

The protein reacts with tartate and cupric sulfate in an alkaline solution, resulting in the formation of a tetradentate copper-protein complex and reducing the Folin-Ciocalteu Reagent. As shown in Figure 2, the blue colored, water-soluble product can be determined at 750 nm. For protein detection, the linearity range is 1-1,500 μg/ml.

If protein probes are measured in microplates, the Modified Lowry Protein Assay can also be carried out in small volumes.

Figure 2. Absorbance scan (300-1,000 nm) of Modified Lowry Protein Assay reagent with (▬) and without (▬) BSA on Infinite M200 reader.

BioRad protein (Bradford) assay

This application uses a dye-binding assay called BioRad Protein Assay. The assay is based on the method devised by Bradford [3]. The Coomassie® Brilliant Blue G-250 dye binds to basic and aromatic amino acid residues, particularly to arginine.

The binding causes a shift of the absorbance maximum of the dye from 465 to 595 nm, as shown in Figure 3. A typical standard curve for measuring protein concentration ranges between 20 and 140 μg/ml if prepared with BSA.

Figure 3. Absorbance scan (300-900 nm) of BioRad Protein assay reagent with (▬) and without (▬) BSA on Infinite M200 reader.

Users can perform the Bradford Assay as a microassay procedure with a linearity range of 1.2-10 μg/ml BSA. This procedure is well suited for measurements in microtiter plates using small volumes.

Material and methods

Instruments

  • Infinite M200, Quad4 monochromator detection system (Tecan Austria)
  • Infinite F200, filter-based detection system (Tecan Austria)

Microplates

  • UV-cuvette micro (Brand, Germany)
  • 96-well flat bottom transparent microplate (Greiner Bio-One, Germany)

Reagents and assay performance

Reagents

  • BCA Protein Assay (Pierce, Illinois): the kit includes BCA Reagents A and B, and BSA protein standard (2 mg/ml)
  • Modified Lowry Protein Assay (Pierce, Illinois): the kit includes Modified Lowry Protein Reagent, BSA protein standard (2 mg/ml), and 2N Folin-Ciocalteu reagent
  • Bio-Rad Protein Assay (Bio-Rad, California): the kit includes protein assay dye reagent concentrate, lyophilized BSA protein standard

Assay protocol

The protein standards, working solutions, etc. for all assays were prepared according to the manufacturers’ recommendations.

BCA protein assay

The BCA Protein Assay is described by two different procedures: one for microplates and the other for cuvettes, both within a working range of 20-2,000 μg/ml.

  • For the microplate protocol, 200 μl BCA working reagent and 25 μl sample are mixed together
  • For the cuvette protocol, 100 μl sample is mixed with 2 ml BCA working reagent
  • If cuvettes are used, it is enough to mix 50 μl sample with 1 ml BCA working reagent

For both the microplate and cuvette procedures, samples are incubated for at 37 °C for 30 minutes and cooled to room temperature before taking the absorbance measurements (Table 2).

Table 2. Measurement parameters and settings for the BCA Protein Assay on Infinite F200 and Infinite M200

Infinite F200 (microplate)

Parameter

Setting

mode

absorbance

wavelength

562 nm

bandwidth

10 nm

number of readings

25

Infinite M200 (microplate, cuvette)

Parameter

Setting

mode

absorbance

wavelength

562 nm

bandwidth

9 nm

number of readings

25

 

Modified Lowry protein assay

There are two different ways in which the Modified Lowry Protein Assay can be carried out: the cuvette protocol is suitable for high volumes of protein samples, while for small volumes of unknown protein a microplate procedure can be used.

The working range for the two procedures is about 1-1,500 μg protein/ml. A multichannel pipettor is recommended for this type of assay, where precise and accurate pipetting has to be carried out on time.

In the case of microplate assays, 40 μl sample and 200 μl Modified Lowry Reagent are mixed together, and the plate is immediately shaken for exactly 30 seconds and incubated for 10 minutes at room temperature.

20 μl of Folin-Ciocalteu Reagent is added and the plate is again shaken for another 30 seconds and incubated at room temperature for 30 minutes, before measuring absorbance at 750 nm.

In the cuvette assays, each sample of 200 μl is pipetted into cuvettes, and 1 ml Modified Lowry Reagent is added after an interval of 15 seconds. After mixing each cuvette well, it is incubated for 10 minutes and 100 μl Folin-Ciocalteu Reagent is added using the same time regime as described earlier.

After mixing the cuvettes, they are incubated at room temperature for 30 minutes, prior to the absorbance measurement at 750 nm (Table 3).

Table 3. Measurement parameters and settings for the Modified Lowry Protein Assay on Infinite F200 and Infinite M200

Infinite F200 (microplate)

Parameter

Setting

mode

absorbance

wavelength

750 nm

bandwidth

10 nm

number of readings

25

Infinite M200 (microplate, cuvette)

Parameter

Setting

mode

absorbance

wavelength

750 nm

bandwidth

9 nm

number of readings

25

 

BioRad protein (Bradford) assay

The sample and dye reagent volumes vary and are protocol-dependent, and Therefore should be determined according to the manufacturer’s manual. Generally, the unknown protein samples and the standard samples are pipetted into the wells and the respective cuvettes.

The corresponding volume of dye reagent is added and then incubated at room temperature for 5 minutes. Sample absorbance is measured at ~595 nm, as seen in Table 4.

Table 4. Measurement parameters and settings for BioRad Protein (Bradford) Assay on Infinite F200 and Infinite M200

Infinite F200 (microplate)

Parameter

Setting

mode

absorbance

wavelength

590 nm

bandwidth

10 nm

number of readings

25

Infinite M200 (microplate, cuvette)

Parameter

Setting

mode

absorbance

wavelength

595 nm

bandwidth

9 nm

number of readings

25

 

Results

BCA protein assay

When measured on the Infinite F200 or Infinite M200, the BCA Protein Assay can be carried out in 96-well microplates using the standard assay procedure, as shown in Figure 4.

When using the Infinite 200 series of instruments, the measured sensitivity for protein detection is roughly 10 μg/ml. In addition, this assay was carried out with its original protocol in cuvettes determined on the Infinite M200 instrument.

The results of the cuvette and microplate measurements are illustrated in Figure 4. Reliable and comparable results are obtained using microplates or cuvettes.

Figure 4. BCA Protein Assay measurements in cuvette using an Infinite M200 (▬) and in 96-well microplate measured on Infinite F200 (▬) and Infinite M200 (▬). The linearity range is 25-2,000 μg/ml.

Modified Lowry protein assay

The Modified Lowry Protein Assay was carried out in both cuvettes and 96-well microplates. Almost identical results may be acquired using microplates on the Infinite F200 and Infinite M200.

The cuvettte measurements show several higher absorbance values than microplate-based measurements. This is because handling cuvettes takes a longer time than measuring a 96-well plate, and the color development also increases during this time (Figure 5).

Figure 5. Modified Lowry Protein Assay measurements in cuvette using an Infinite M200 (▬) and in 96-well microplate measured with Infinite F200 (▬) and Infinite M200 (▬).

Using an Infinite M200 or Infinite F200 instrument, the sensitivity of the Modified Lowry Protein Assay is measured to be about 2 μg/ml (Table 5).

Table 5. Sensitivity [μg protein/ml] of the BCA, Modified Lowry and BioRad (Bradford) Protein Assays measured on Tecan’s Infinite 200 instruments. Linearity ranges of the different protocols are given in μg/ml

Assay

BCA

Lowry

Bradford

Protocol [μg/ml]

25-2,000

26-1,500

8-80

Sensitivity [μg/ml]

10

2

1

 

 

BioRad protein (Bradford) assay

For low protein concentrations, differing in dye/sample ratio, the following microassay protocols are available:

  • a cuvette protocol for protein concentrations from approximately 1-12 μg/ml
  • a microplate protocol for protein concentrations ranging from 8 to 80 μg/ml

On performing the Bradford microassays in cuvettes and microplates, the sensitivity is measured to be about 1 μg/ml protein.

Figure 6. BioRad Protein (Bradford) microassays measured on Infinite F200 (▬) and on Infinite M200 (▬) in 96-well microplates, and on Infinite M200 (▬) using cuvettes.

Discussion

Tecan’s Quad4 monochromator-based Infinite M200 and filter-based Infinite F200 instruments were used to compare three different protein quantification assays.

With regard to the BCA assay, the standard procedure was used for microplates and cuvettes, though this protocol is considered to be unsuitable for microplates. The two instruments are capable of detecting protein concentrations of about 10 μg/ ml.

For lower protein concentrations, the Modified Lowry Protein Assay is useful as it uses a single protocol for all dilutions with estimated sensitivity of 2 μg/ml. However, this assay is very laborious, and sample handling and pipetting become more difficult when the sample numbers increase.

Therefore, for this experiment, the Infinite 200 injector system was used to carry out the Modified Lowry Protein Assay in 96-well plates. The application note ’Protein quantification on Infinite 200 with injectors’ (#395179) contains details and instrument settings.

The BioRad Protein (Bradford) microassay protocols can be employed for sensitive protein detection. Measurements can be done in cuvettes and 96-well plates, protein concentrations of up to 500 μg/ml can be determined using the standard assay protocol.

Conclusion

The assay selected for protein quantification is based on the composition of the sample (detergents, reducing agents, salts, and buffer), the quantity of protein available, and the nature of the protein. Limitations occur on the instrument side, if more samples have to be measured and the reader is able to read only cuvettes.

Such conditions require a higher amount of protein in the sample. However, these issues can be resolved by using the Infinite 200 series of microplate readers from Tecan. These readers provide high sensitivity and high flexibility.

As shown in this article, microplates can be used to measure small quantities of protein in small volumes (Table 5). In addition, the Infinite M200 monochromator reader is capable of measuring protein samples in both microplates and cuvettes.

Absorbance maxima of unknown substances can be detected, since the Infinite M200 can also carry out absorbance scans. This was shown for the protein assays described in this article, when an absorbance scan of the colored complexes was performed, with and without BSA.

List of abbreviations

  • BCA     bicinchoninic acid
  • BSA      bovine serum albumin
  • Cu        copper

Literature

  1. Smith, P.K., et al.: Measurement of protein using bicinchoic acid. Anal Biochem., 150, 76-85, 1985
  2. Lowry, O.H. et al.: Protein measurement with Folin Phenol Reagent. J. Biol. Chem., 193, 265-275, 1951
  3. Bradford, M.M.: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding. Anal. Biochem., 72, 248-254, 1976

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.

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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: May 16, 2017 at 2:25 PM

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