Protein Quantification by the Lowry Method
I. Introduction
The Lowry method is one of the most widely used assay to estimate protein content in
biological samples. This method is based on the biuret reaction: under alkaline conditions,
the divalent copper ion forms a complex with peptide bonds in proteins and is reduced to
a monovalent ion. Subsequently, monovalent copper ion and the radical groups of tyrosine,
tryptophan, and cysteine in proteins react with Folin-Ciocalteu reagent to produce a blue-
green color that can be detected between 650 nm and 750 nm. The method is sensitive
down to about 0.01 mg of protein/mL. Concentrations of protein recommended for
quantitative with Lowry method are in the range 0.01–1.0 mg/mL. It would be important
to underline that Lowry method is sensitive to the amino acid composition (contents of
tyrosine, tryptophan, and cysteine) of the protein and absolute concentrations cannot be
obtained.
II. Materials
- Beaker, meansuring cylinder, mortar and pestle, Erlenmeyer (conical) flask, volumetric
flask, glass funnel, water bath.
- Solution A: 2% (w/v) Na2CO3 in NaOH 0.1N.
- Solution B: 0.5% (w/v) CuSO4.5H2O in 1% sodium citrate or 1% sodium potassium
tartrate.
- Solution C: The mixture of solution A and B with ratio 50:1
- 0.1 N NaOH.
- Folin-Ciocalteu reagent
- BSA stock solution (1 mg/mL).
- Filter paper.
- pH paper.
III. Procedure
3.1. Preparation of Standard Curve
- Prepare Standards in glass tubes as indicated in the Table 1. For greater accuracy, run
this step in duplicate.
Table 1. Preparation of BSA Standard Solutions
Tube 1 2 3 4 5 6
1 mg/mL 0 0.1 0.2 0.3 0.4 0.5
BSA (mL)
H2O (mL) 0.5 0.4 0.3 0.2 0.1 0
BSA
concentration
Ci (mg/mL)
- To each tube add 5 mL Solution C, vortex, wait for 10 min.
- To each tube add 0,5 mL Folin-Ciocalteu reagent, vortex immediately, wait for 20 min
- Measure A750nm. Ideally, the absorbances should be between 0.1 and 0.5. Note: Read
carefully the instruction for use of the spectrophotometer before use and set the
spectrophotometer to zero by using blank solution (0 mg/mL BSA).
- Complete the Table 2
Table 2. Absorbance versus BSA concentration
BSA
concentration
Ci (mg/mL)
A750nm
- Prepare the standard curve by plotting the absorbance value for each BSA standard vs.
its concentration in mg/mL. Use the following link for tutorial:
https://www.youtube.com/watch?v=KeTFzJUG_rA
3.2. Sample Preparation
- Weigh about 0.3 g of sample in a small mortar.
- Take 20 mL NaOH 0.1 N in a beaker.
- Pipette 1 mL NaOH 0.1 N into the mortar to moist the sample.
- Grind the sample using a pestle.
- Transfer the homogenate to 100 ml conical flask.
- Rinse the mortar and pestle by using the remaining of NaOH 0.1 N and transfer to the
100 ml conical flask.
- Incubate at 100oC in a water bath for 15 min.
- Cool the conical flask with tap water.
- Adjust the pH of solution to 7-8 with HCl 0.1 N using pH paper.
- Tranfer the homogenate to a 100 mL volumetric flask.
- Rinse the conical flask thoroughly with distilled water and transfer to the volumetric
flask.
- Use a pipette or dropper to fill the volumetric flask with water, using the meniscus of
the solution and the line on the flask to determine the endpoint.
- Seal the volumetric flask and invert it to thoroughly mix the solution.
- Filter the solution following the link: https://www.youtube.com/watch?v=or8lhShqxZg
3.3. Lowry assay
Blank sample
- Add 0.3 mL of water to a glass tube.
- Add 3 mL Solution C to the tube, vortex and wait for 10 min.
- Add 0.3 mL Folin-Ciocalteu reagent, vortex immediately, wait for 20 min.
- Set the spectrophotometer to zero by using blank solution.
Test sample
- Add 0.3 mL of filtered sample to a glass tube.
- Add 3 mL Solution C to the tube, vortex and wait for 10 min.
- Add 0.3 mL Folin-Ciocalteu reagent, vortex immediately, wait for 20 min.
- Measure A750nm. Ideally, the absorbances should be between 0.1 and 0.5.
- Determine the concentration of protein from the standard curve and the protein content
of the sample.
