Spectrophotometric Protein Assays Spectroscopic methods are routinely used to determine the concentration of proteins in a solution. These include measurement of the protein’s intrinsic UV absorbance...

Spectrophotometric Protein Assays

Spectroscopic methods are routinely used to determine the concentration of proteins in a solution. These include measurement of the protein’s intrinsic UV absorbance and four methods that generate a protein-dependent color change: the Biuret, Lowry, Bradford and the Smith copper/bicinchronic acid (BCA) assay. This exercise introduces students to four of the five methods of determining protein concentrations: absorbance at 280 nm, and the Biuret, Lowry, and Bradford assays. Bovine serum albumin (BSA) is typically used as a reference protein standard so you will determine calibration curves for solutions of BSA at 0.1, 0.2, 0.3, 0.5, and 0.7 mg/mL for Abs₂₈₀, Lowry, and Bradford. The Biuret is measured at higher concentrations of 1, 2, 4, and 6 mg/mL in water. The assay procedures are as follows:

A₂₈₀:
Measure the absorbance of 1 mL at 280 nm.

Lowry:
Mix 0.25 mL of protein with 2.5 mL of Lowry reagent 1. After 10 minutes, add 0.25 mL of Lowry reagent 2 and mix well immediately. After 30 minutes, measure the absorbance at 750 nm (if you're using a Spectronic 20 with a normal phototube, 750 may be too long; 600 nm gives lower absorbances but works okay).

Bradford:
Mix 0.25 mL of protein with 2.5 mL of Bradford reagent and measure the absorbance at 595 nm.

Biuret:
Mix 0.50 mL of protein with 2.50 mL of biuret reagent and measure the absorbance at 540 nm.
Perform all tests in duplicate. All series should include a zero protein (water) tube (reagent blank). You can zero the Spectronic 20’s to the reagent blanks. This is especially important with the Bradford assay, for which the reagent blank has quite a high absorbance (>0.5). Two other practical concerns with the Bradford assay are its great sensitivity to detergent (tubes must be rinsed very well) and the fact that it stains the cuvettes blue.

After

plotting

your data on separate graphs for each assay, evaluate each method by the criteria of(1) convenience (how easy is it to do?),(2) sensitivity (how well does it detect small amounts of protein; for example, what mass of protein is needed to give an absorbance of 0.1?), (3) generality (how consistent are the results among the two different proteins?), and(4) linearity (does it give a straight line plot of absorbance vs. protein?).


Reagents


Biuret Reagent:
Add, with stirring, 300 mL of 10% (w/v) NaOH to 500 mL of a solution containing 0.3% copper sulfate pentahydrate and 1.2% sodium potassium tartrate, then dilute to one liter. The reagent is stable for a few months but not a year. Adding one gram of potassium iodide per liter and storing in the dark makes it stable indefinitely.
The reagent can be used either qualitatively or quantitatively. In a typical reaction, one volume of sample is mixed with two to five volumes of reagent; the optimal ratio depends on the maximum protein concentrations you want to be able to resolve. The presence of protein gives a violet color with maximum absorbance around 550-555 nm; we typically read the absorbance at 540 nm.

Bradford's Reagent:
The original published recipe [see
Analyt. Biochem.
72, 248-254 (1976)] calls for dissolving 100 mg Coomassie Blue G-250 in 50 mL of 95% ethanol, add 100 mL of 85% phosphoric acid, and dilute to one liter. The reagent needs to be filtered at least once and perhaps more, since it seems to precipitate dye over time. "Bradford reagents" are available commercially that use more stable formulations.*
* Internet sites report that Sigma/Aldrich's formula uses 40 mL of methanol (final 4%) in place of ethanol and about 120 mL of phosphoric acid (final 10%).
To quantify protein, mix 0.25 mL of sample with 2.5 mL of Bradford reagent. After 5 minutes, measure the absorbance at 595 nm. One disadvantage to the reagent is that it gives a high blank which may affect subsequent readings because some reagent adheres to the cuvette. Another is that it is very sensitive to the presence of detergent, either from poorly-rinsed glassware or, heaven forbid, in the event you are studying detergent-solubilized membrane proteins.

Lowry Reagents:Reagent 1: Mix one volume of reagent B (0.5% copper sulfate pentahydrate, 1% sodium or potassium tartrate) with 50 volumes of reagent A (2% sodium carbonate, 0.4% NaOH). Both reagents A and B are supposed to be stable for a long time but precipitation in reagent B could occur over time that can to be remedied by adding a little NaOH.
Reagent 2: Dilute commercial Folin-Ciocalteu phenol reagent with an equal volume of water. Stable for a few days or weeks.
To quantify protein, mix 0.25 mL of protein with 2.5 mL of Lowry reagent 1. After 10 minutes, add 0.25 mL of Lowry reagent 2 and mix well immediately. After 30 minutes, measure the absorbance at 750 nm (if you're using a Spectronic 20 with a normal phototube, 750 is too long; 600 nm gives lower absorbances but works okay).

Reference Protein Behavior in Various Assays

Comparison of Protein Assays	Estimated Protein Concentration (mg/mL) for actual Conc. of 10 mg/mL
Protein	MW (x103)	pI	Abs1% 280nm	Biuret	Lowry	Bradford
Alcohol Dehyrogenase (yeast)	141(4)	5.4	12.6	5.8	5.0	7.8
Alkaline phophatase (E. coli)	89(2)	4.5	7.2
a-amylase	50+	-	26	6.8	6.0	8.3
Asparaginase (E. coli)	136 (4)	7.1
Bovine Serum Albumin	66.2	5.2	6.6, 5.8	9.7	8.4	21.1
Carbonic anhydrase	29.5	5.9	18	8.8	8.9	15.2
Catalase	250 (4)	5.4	6.7	7.6	6.3	9.7
a-Chymotrypsin (bovine)	25	9.1, 5.2	20.4	9.4	11.6	9.5
Cytochrome c (bovine)	12.3	10.6	19.2, 18.4	25.7	11.3	26.2
Ovalbumin	45	4.6	7.5, 5.1	10.2	10.1	11.6
Fibrinogen (bovine)	330	13.6	6.2	7.3	7.8
Gamma globulin (bovine)	155 (2+2)	6.6	13.8	10.0*	10.0*	10.0*
Gamma globulin (rabbit)	155 (2+2)	6.6	13.8	9.4	11.8	8.9
b-Galactosidase	540 (4)	20.9	9.5	9.9	7.9
Hemoglobin (bovine)	66 (4)	6.8	16.2	8.3	19.9
Lipase (porcine pancrease)	45-50	4.9, 5.0	30, 13.3
Lysozyme (chicken egg white)	14.4	11	26.4	10.4	12.6	7.4
Myoglobin	17	7.0	17.8	13.7	7.9	24.9
Ovomucoid	28	7.8	8.3	5.1
Papain	23	9.6	25
Pepsin	35	1.0	14.7	9.8	12.4	3.4
Ribonuclease (bovine pancrease)	13.7	9.5	7.3	11.8	15.9	9.9
Trypsin inhibitor (soybean)	21.5	4.6	9.5, 6.0	9.1	10.3	10.1
Transferrin (bovine)	77	11.2	8.5	9.0	12.6
Trypsin (bovine pancrease)	23.8	10.5	14.3	11.4	15.5	5.5
Thyoglobulin	669	7.7	8.2	9.3
Average		10.1	9.5	11.8
Standard Deviation		4.2	2.9	6.3