Procedure

UV absorbance Spectroscopy:

For measuring the concentration of a protein with an unknown extinction coefficient using a protein standard, follow these steps:

1. Add blank buffer to a clean quartz cuvette and use it to zero the spectrophotometer.
2. Either use a fresh, identical cuvette or replace the buffer with the sample. Measure the absorbance at 280 nm. If the signal is outside the linear range of the instrument (typically an absorbance greater than 2.0), dilute the protein in buffer and remeasure.
3. After measuring the sample, remeasure the blank buffer to correct for any instrument drift.
4. Determine the unknown concentrations from the linear standard response.
5. Read the absorbance of the test protein versus a suitable control at 280 nm.
6. A very rough approximation for the proteins is 1 absorbance unit is equal to 1 mg/mL.

For example,

• Protein A280 (for 1 mg/mL); IgG 1.35 ; IgM 1.2; BSA 0.7

Protein Solutions Contaminated with Nucleic Acids

1. Read the absorbance versus a suitable control at 280 and 260 nm, or 280 and 205 nm.
2. Calculate the approximate concentration using one of the equations below:

   Protein concentration (in mg/mL) = (1.55 × A280) − (0.76 × A260)

Light scattering from turbid protein solutions

The light scattering from turbid protein samples imposes an error in measuring the absorbance at 280 nm. The apparent increase in absorbance gives an overestimated concentration of the protein. The suspended particles can reduce the light that reaches the detector and cause an erroneous increase in absorbance.

Filter and centrifugation can ensure accurate measurements for such turbid samples (with particles).

Dye-based protein assays

Reagent Requirement:

1. Dissolve 100 mg of Coomassie Brilliant Blue G-250 in 50 ml of 95% ethanol.
2. Add 100 ml of 85% phosphoric acid while stirring continuously.
3. Once the dye has dissolved, dilute the solution to 1 liter with water.
4. The reagent is stable for up to a month at room temperature. For long-term storage, keep it at 4°C. If precipitation occurs, filter the solution before use.

Procedure:

1. Prepare standards in the range of 100–1500 µg/ml using a Bradford-compatible buffer. For more dilute samples, increase the ratio of sample to reagent volumes (Micro Bradford assay: 1–25 µg/ml). Note that if the ratio of the sample to dye is too high, the pH of the reaction mixture may increase, leading to higher background responses.
2. Add the standard and unknown samples to disposable cuvettes. Use plastic disposable cuvettes and microplates, as the dye tends to stick to various surfaces.
3. Allow the Bradford reagent to warm to room temperature. Add 1 ml of the dye solution to 25 µl of the protein sample, mix, and incubate for 10 minutes at room temperature.
4. Measure the absorbance at 450 nm and 595 nm. For filter-based instruments, a range from 570 nm to 610 nm can be used without significant loss of assay performance.
5. Plot either the 595 nm data or, for improved precision at lower response values, the ratio of 595 nm/450 nm. Fit the standard response curve to a polynomial response, from which unknown protein concentrations can be calculated.

Caution:

• Watch for the ratio of the sample to dye, if it is too high, the pH of the reaction mixture could increase leading to higher background responses.
• Allow the Bradford reagent to warm to room temperature. Add 1 ml of the dye solution to 25 µl of the protein sample, mix and incubate for 10 min at room temperature.

Precaution:

1. This assay is relatively accurate for most proteins, except for small basic polypeptides such as ribonuclease or lysozyme. It is also hampered by detergent concentrations greater than 0.2% (e.g., Triton X-100, SDS, NP-40).
2. Results with commercial Bradford reagent from Bio-rad gives consistent results.
3. Bradford assay is sensitive to glycosylated proteins.
4. If proteins are not solubilized, the reaction can be supplemented with 1 M NaOH at 5–10% (v/v) to aid solubilization.

Lowry (Alkaline Copper Reduction Assays) (Range: 5–100 µg)

Reagents

Folin and Ciocalteu’s Reagent

The preparation of this reagent has been described by Lowry et al. (1951). It can also be obtained from commercial sources such as Sigma. To prepare, mix 10 ml of Folin–Ciocalteu’s Phenol reagent with 50 ml of water.

Copper Sulfate Reagent

Dissolve 100 mg of CuSO₄·5H₂O and 200 mg of sodium tartrate in 50 ml of water. Separately, dissolve 10 g of sodium carbonate in 50 ml of water, then slowly pour this solution into the copper sulfate solution while mixing. Prepare fresh daily.

Alkaline Copper Reagent

Mix one part copper sulfate solution, one part 5% SDS (w/v), and two parts 3.2% sodium hydroxide (w/v). This solution can be stored at room temperature for up to 2 weeks. Discard if a precipitate forms.

Procedure:

1. Add 1 ml of the alkaline copper reagent to 1 ml of sample and protein standards 5–100 mg/ml, mix, and allow to stand for 10 min.
2. Add 0.5 ml of Folin–Ciocalteu’s reagent mix, vortex thoroughly, and incubate for 30 min.
3. After incubation vortex again and measure the absorbance at 750 nm. Absorbance can be read from 650 to 750 nm depending on the availability of appropriate filters (microplate readers), or if the signal is too high, without significant loss in assay performance.

Modified Lowry (with SDS)

1. Before the assay, dissolve 10 g of Na2CO3 in 500 mL of distilled water. Dissolve 0.5 g of CuSO4·5H2O and 1 g of Na tartrate in 500 mL of distilled water. Place the copper/tartrate solution on a magnetic stirrer, and slowly add the carbonate. Store with refrigeration. This solution is stable for >1 yr.
2. Reagent A: Combine 1 volume of the copper/tartrate/carbonate solution to 2 volumes of 5% SDS, and 1 volume of 0.8 M NaOH. This is stable at room temperature for 2 wk.
3. “Reagent B.” Combine 1 volume of the 2 N Folin–Ciocalteu phenol reagent with 5 volumes of distilled water. Store in an amber bottle at room temperature. This solution is stable for months.
4. Samples (of 5–100 μg) should be adjusted to 1 mL by adding water. Prepare standards of BSA containing 100, 50, 25, and 12.5 μg/mL.
5. Add 1.0 mL of Reagent A to each protein sample. Mix and incubate for 10 min at room temperature.
6. Add 0.5 mL of Reagent B and mix immediately. Incubate for 30 min at room temperature.
7. Read the absorbance at 750 nm. Prepare a standard curve, and compute the protein concentration.

Bicinchoninic Acid (BCA) (Range: 0.2–50 µg)

Reagents:

Reagent A: 1 g sodium bicinchoninate, 2 g Na2CO3, 0.16 g sodium tartrate, 0.4 g NaOH, and 0.95 g NaHCO3, made up to 100 ml and the pH adjusted to 11.25 with either solid or concentrated NaOH.

Reagent B: 0.4 g CuSO4·5H2O dissolved in 10 ml water. Both reagent A and B are stable indefinitely at room temperature. The working solution is prepared by mixing 100 parts of reagent A with two parts reagent B to form a green solution that is stable for up to a week.

Procedure:

1. Cuvette analysis can be performed with 50–150 ml of protein and 3 ml of BCA working reagent, whereas microplate assay can use 25 µl of protein and 200 µl of BCA working reagent, that is a lower reagent to protein ratio.
2. Incubate the sample and standards 5–250 µg/ml at either 37 or 60°C for 30 min (longer incubations at 37°C will improve protein-to-protein variability) and allow the sample to equilibrate to room temperature before reading. Microplates should be covered during incubation to avoid evaporation of the sample.
3. Measure absorbance at 562 nm, for filter-based plate readers wavelengths in the range of 540–590 nm can be used instead without a significant loss in assay performance.
4. A microplate-based BCA assay can be used to improve the sensitivity of the procedure (1–25 µg/ml). The microplate-based assay uses a more concentrated working solution and can be prone to precipitation; again, commercial sources of this modified BCA assay are available (Pierce). DTT interferes in the BCA assay as this reagent is sensitive to either copper chelators (e.g., EDTA) or reagents that can also reduce Cu2þ (e.g., DTT).