Estimation of protein by Lowry's method
Alkaline copper binds peptide bonds, then Folin-Ciocalteu reagent is reduced to a blue colour read at 660 nm and compared with a BSA standard graph.
Aim and principle
Aim: To estimate the amount of protein in the given solution by Lowry's method, using bovine serum albumin (BSA) as the standard. Principle: The colour forms in two steps. First, in alkaline solution, Cu2+ binds the peptide bonds (the biuret reaction) and is reduced to Cu+. Second, the Folin-Ciocalteu reagent (phosphomolybdate-phosphotungstate) is reduced by the copper-protein complex and by the tyrosine and tryptophan side chains to an intense blue (molybdenum blue). The depth of blue, read at 660 nm (some manuals use 750 nm, as in Lowry's original method; use the one in your manual), is proportional to the protein present within the working range (Beer-Lambert law). A standard graph of absorbance against µg BSA gives the protein in the sample. Alternative: the biuret method uses the copper step alone (violet, read at 540 nm); it is less sensitive (milligram amounts) but less affected by the amino acid make-up.
Requirements
| Reagent or item | Made of | What it does |
|---|---|---|
| Reagent A | 2% sodium carbonate in 0.1 N NaOH | Gives the alkaline medium |
| Reagent B | 0.5% copper sulphate in 1% sodium potassium tartrate | Supplies Cu2+; tartrate keeps it dissolved |
| Reagent C | 50 mL A + 1 mL B, mixed fresh on the day | Alkaline copper reagent (the biuret step) |
| Reagent D | Folin-Ciocalteu reagent diluted 1:1 with water just before use | Reduced to the blue colour |
| BSA standard | Stock: 50 mg BSA in 50 mL water (1 mg/mL). Working: 10 mL stock made up to 50 mL (200 µg/mL) | For the standard graph |
| Apparatus | Colorimeter or spectrophotometer (660 nm, red filter), cuvettes, test tubes, graduated pipettes or micropipettes, timer, graph paper | Reading and plotting absorbance |
| Safety | Gloves and goggles | Folin reagent is acidic and corrosive; pipette with a bulb |
Procedure
Volumes follow the common protocol. Follow the exact volumes in your lab manual.
- 1Pipette 0.2, 0.4, 0.6, 0.8 and 1.0 mL working standard (40-200 µg BSA) into labelled tubes.
- 2Pipette 0.2 and 0.4 mL of the (diluted) unknown into two more tubes. Make every tube up to 1 mL with water; 1 mL water alone is the blank.
- 3Add 5 mL reagent C to every tube; mix and leave for 10 minutes at room temperature.
- 4Add 0.5 mL reagent D to each tube and mix at once.Folin reagent breaks down quickly in alkali
- 5Leave in the dark at room temperature for 30 minutes.Blue colour develops
- 6Set the colorimeter to zero with the blank at 660 nm and read every tube.
- 7Plot absorbance (y-axis) against µg protein (x-axis) and read the unknown from the graph.Use a reading that falls inside the standard range
Observation table
| Tube | Standard or sample (mL) | Protein (µg) | Water (mL) | Reagent C (mL) | Reagent D (mL) | Absorbance at 660 nm |
|---|---|---|---|---|---|---|
| Blank | 0 | 0 | 1.0 | 5.0 | 0.5 | 0.00 |
| S1 | 0.2 | 40 | 0.8 | 5.0 | 0.5 | 0.12 |
| S2 | 0.4 | 80 | 0.6 | 5.0 | 0.5 | 0.24 |
| S3 | 0.6 | 120 | 0.4 | 5.0 | 0.5 | 0.36 |
| S4 | 0.8 | 160 | 0.2 | 5.0 | 0.5 | 0.48 |
| S5 | 1.0 | 200 | 0 | 5.0 | 0.5 | 0.60 |
| Test | 0.4 (diluted unknown) | From graph | 0.6 | 5.0 | 0.5 | 0.39 |
Worked example: protein in the given solution
The unknown protein solution was diluted 1 mL to 10 mL with water, and 0.4 mL of the diluted solution was taken. Its absorbance at 660 nm was 0.39. The BSA standards gave a straight line (40 µg → 0.12 … 200 µg → 0.60). Find the protein in mg/mL and g/100 mL of the original solution.
- Slope of the standard line = 0.60 ÷ 200 µg = 0.003 absorbance per µg (check: 0.12 ÷ 40 = 0.003).
- Protein in the test tube = 0.39 ÷ 0.003 = 130 µg (the value you read off the graph).
- Protein in 1 mL of the diluted solution = 130 µg ÷ 0.4 mL = 325 µg/mL.
- Undo the dilution (1 to 10): 325 × 10 = 3250 µg/mL = 3.25 mg/mL in the original.
- Per 100 mL = 3.25 mg × 100 = 325 mg = 0.325 g/100 mL.
- Formula: protein (mg/mL) = (µg from graph ÷ mL of sample taken) × dilution factor ÷ 1000. Real points scatter a little; draw the best straight line.
The given solution contains 3.25 mg protein per mL, i.e. 0.325 g/100 mL (as BSA equivalent).
Precautions and result
- Make reagent C fresh, and add reagent D quickly with immediate mixing, because Folin reagent breaks down within seconds in alkali.
- Keep the timing the same for every tube (10 minutes after C, 30 minutes after D).
- Dilute the sample so its absorbance falls inside the standard range; never extend the line beyond 200 µg.
- Avoid interfering substances (ammonium sulphate, Tris, phenols, thiol reagents, detergents); remember the result is in BSA equivalents.
- Gloves and goggles; use a bulb or micropipette, never your mouth; wipe cuvettes clean and dry.
- Result: The amount of protein in the given solution is ____ mg/mL (____ g/100 mL).
Lowry's method measures small amounts of protein and is used in research to find protein in food and plant extracts and to express enzyme activity per mg protein. In the clinic, serum total protein (about 6-8 g/dL) and albumin (about 3.5-5 g/dL) are measured by the related biuret and dye methods; low values point to protein-energy malnutrition (kwashiorkor), liver disease or protein loss, so they are part of nutritional assessment.
Short answer: “What is the role of Folin-Ciocalteu reagent in Lowry's method?” It is reduced by the copper-peptide complex and by tyrosine and tryptophan to an intense blue colour, which is read at 660 nm.
Short answer: “Why is BSA used as the standard?” It is a pure, stable, soluble and inexpensive protein, so known amounts give a reliable standard graph. Results are expressed as BSA equivalents.
Structured question: “Explain the principle of Lowry's method and how protein is calculated from the standard graph.” Two-step colour: Cu2+ binds peptide bonds in alkali; Folin-Ciocalteu is reduced to blue by the copper complex and by tyrosine and tryptophan; read at 660 nm. Plot absorbance of 40-200 µg BSA; read µg for the sample; divide by the volume taken and multiply by the dilution. Example: 0.39 → 130 µg in 0.4 mL of a 1-in-10 dilution = 3.25 mg/mL.
Copper first, Folin second, 660 to read: 5 mL C, wait 10; 0.5 mL D, wait 30.