How to Quantify Proteins

Protein Quantification Methods

While there are an abundance of options to choose from when it comes to protein quantification, three assays are used most often—largely because of convenience, minimal reagents required, and relatively low cost, easy-to-access instruments (e.g., spectrophotometer).

How to Quantify Proteins Using BCA

The BCA assay is one of the most widely used protein assays because it is sensitive and easy to perform. Mechanistically, it involves two reactions: 1) the Biuret reaction where cupric ions are reduced by proteins under alkaline conditions, and 2) the chelation of BCA with the Cu+ ion. The second reaction changes the color of the sample from blue to purple and produces a stable BCA-copper complex that absorbs light at 562 nm.

Reagents

• Solution A: 1 g sodium bicinchoninate (BCA), 2 g sodium carbonate, 0.16 g sodium tartrate, 0.4 g NaOH, and 0.95 gm sodium bicarbonate, brought to 100 ml with distilled water (Adjust the pH to 11.25 with 10 M NaOH)
• Solution B: 0.4 g cupric sulfate (pentahydrate) in 10mL distilled water
• Standard working solution (stable for 1 week and should be green): 50:1 Solution A: Solution B

Protocol

1. Prep the protein samples to a concentration of 0.2-50 µg/µL
2. Add 1 ml of standard working solution to each sample and vortex
3. Incubate for 30 minutes at 60°C
4. Cool the samples
5. Measure the absorbance at 562 nm

Determine your protein quantity by comparing your measured absorbance to a previously prepared standard curve of absorbance versus micrograms of protein.

How to Quantify Proteins Using Bradford

The Bradford assay is a popular method for quantifying proteins that relies on the Coomassie brilliant blue G-250 dye binding proteins (mainly arginine, tryptophan, tyrosine, histidine, and phenylalanine residues) in solution, resulting in a color change from brown to intense blue. You can determine your protein concentration by measuring the absorbance of the solution at 595 nm using a spectrophotometer.

Reagents

You can use commercially prepared Bradford solution or make your own

• Dissolve 100 mg Coomassie Brilliant Blue G-250 in 50 ml 95% ethanol
• Add 100 ml 85% (w/v) phosphoric acid
• Dilute to 1 liter after the dye completely dissolves
• Filter just before use

The Bradford solution should be a light brown color.

Protocol

1. Dilute your sample if needed
2. Optional: If samples are not readily soluble in Bradford solution (you’ll see blue clumps), add an equal volume of 1 M NaOH to each sample and vortex (gently, but thoroughly so your sample remains in the cuvette). Add NaOH to standards if you perform this step
3. Prepare standards containing a range of 5 to 100 micrograms of protein (albumin or gamma globulin are recommended) in 100 µl volume
4. Add 5 ml dye reagent and incubate at room temperature for 5 minutes
5. Measure the absorbance at 595 nm

Similar to the BCA assay, compare your absorbance to a previously generated standard curve of absorbance versus micrograms protein to calculate the concentration of your original samples.

How to Quantify Proteins Using UV Absorbance

Proteins absorb at 280 nm due to the presence of aromatic amino acids tyrosine and tryptophan, with phenylalanine and disulfide bonds also slightly contributing.

Because the UV absorbance method is simple and can be performed with as little as 0.5 µl, it is often a preferred choice for assessing protein concentration. However, for pure protein samples, you’ll need to know the exact amino acid sequence of the analyzed protein and the associated absorption coefficient.

Protocol

1. Warm up the UV lamp for 15 minutes
2. Set the wavelength to 280 nm
3. Calibrate the UV lamp to zero absorbance using a blank (buffer solution)
4. Measure the absorbance of your protein solution

If you believe you may have a case of nucleic acid contamination, continue with the steps below

5. Adjust the wavelength to 260 nm
6. Calibrate the UV lamp to zero absorbance using a blank (buffer solution)
7. Measure the absorbance of your protein solution

Calculating protein concentration

The concentration for known, pure proteins is mg/ml, %, or molarity depending on the coefficient used. mg/ml = % protein/10 = molarity/protein molecular weight