Introduction

Generally, protein quantitation can be made using a simple UV-Visible spectrophotometer. The V-630 Bio (Figure 1) is a UV-Visible spectrophotometer designed for biochemical analysis. The V-630 Bio includes 6 quantitative methods based on UV absorption spectrophotometry including the Lowry, Biuret, BCA, Bradford, and WST methods.

Table 1 (below) shows the features of the six different quantitation methods. As outlined, a method can be selected by reviewing the sample and quantitation range and the presence of possible contaminants. Five of the analysis methods use a chromogenic reaction. Reagent manufacturers produce chromogenic kits for BCA, Bradford or WST with a protocol explaining the relevant measurement procedures ^*1). Chromogenic reagents for the Lowry and Biuret methods must be prepared by the user. The measurement procedures for the Lowry and Biuret methods differ according to use. For that reason, this application note explains how to create the calibration curves for the Lowry, Biuret and UV absorption analysis methods. Fluctuations in the values depending on the type of proteins and the differences in the concentration ranges calculated as a result of the cell volume ^*2) were also investigated.

*1) BCA method: BCA Protein Assy Kit – by PIERCE
Bradford method: Protein Quantification Kit – Rapid by DOJINDO
WST method: Protein Quantification Kit – Wide Range by DOJINDO

*2) 10 mm rectangular cells (quartz) and disposable cells (Figure 3) were used for the experiments. The optional cell holder accessory enables
the measurement of a sample with a small volume (minimum volume: 100µL)

Table 1. Details of the Protein Quantitation Methods

*BSA: Bovine Serum Albumin

1. UV Absorption Method

Figure 4 illustrates the absorption spectrum of Human Serum Albumin (HSA). Simple UV Absorption spectrophotometry can determine the quantity of proteins in the sample by using the maximum absorption at 280 nm.

1.1 Samples

Bracketed values are for concentrations used with a 10 mm rectangular cell. The other values are concentrations used with a 10 mm rectangular cell and micro cell.

1.2 Measurement Procedures

Measure the absorbance of the protein solution at 280 nm.

Table 2. Results for the UV Absorption Method

2. Biuret Method

Protein solutions turn purple with an absorption maximum of 540 nm when Biuret reagent is added (Figure 5).

The time course of the chromogenic reaction was measured at 540 nm absorption. The absorbance became stable approximately after 60 minutes (Figure 6). Figure 7 shows the spectra of the HSA aqueous solutions that were measured after 60 minutes. The Biuret method determines the quantity by using the absorption maximum at 540 nm after reacting with the sample for 60 minutes.

2.1 Chromogenic Reagent

Biuret reagent: Add 60 mL of 10% NaOH to 100 mL of aqueous solution to dissolve 0.3 g CuSO4 and 1.2 g Rochelle salt, and then add water to make up to a total volume of 200 mL. (The standard reagent can be stored in a polyethylene bottle.)

2.2 Samples

2.3 Measurement Procedures

Add Biuret reagent (2.0 mg) to 500 µL of protein aqueous solution and mix them well. Then react the solution for 60 minutes. Measure the absorbance at the wavelength of 540 nm.

Table 3. Results for the Biuret Method

3. Lowry Method

Protein solutions turn blue with an absorption maximum of 750 nm when Alkline copper solution and Phenol reagent are added to a protein solution (Figure 8).

The time course of the chromogenic reaction was measured at 750 nm absorption. The absorbance became stable after approx. 60 minutes (Figure 9). Figure 10 shows the spectra of the HSA aqueous solutions that were measured after 60 minutes.

The Lowry method determines the quantity by using the absorption maximum of 750 nm after reaction of the sample for 60 minutes.

3.1 Reagents

Alkaline copper solution: Mix 50 mL of 2% Na2CO3 solution ^*3) with 1 mL of 0.5% CuSO4 solution*4) (can only be used for immediate analysis).

2% Na₂CO₃ solution: dissolve anhydrous sodium carbonate (2 g) and caustic soda (0.4 g) in 100 mL water
0.5% CuSO₄ solution: dissolve copper (II) sulfate pentahydrate (50 mg) and potassium sodium tartrate tetrahydrate (0.1 g) in 10mL of water.

Phenol reagent: Dilute commercial solution (2N phenol chemical reagent of Kanto Chemical) to 1N.

3.2 Samples

Same concentrations are used both for the 10 mm rectangular cell and the micro cell of 10 mm.

3.3 Measurement Procedures

Add 2.5 mg of alkaline copper solution to 500 µL of protein solution. Allow to react for 10 minutes after mixing well. Then, add phenol reagent. Rapidly mix and allow the solution to react for 60 minutes. Measure the absorbance at 750 nm.

Table 4. Results for the Lowry Method

Keywords

UV-003, V-630bio, Protein concentration, protein quantitation, colorimetric assay, Bradford, BCA, Lowry, Biuret, 260/280