What is Lowry Protein Assay?
The Lowry Protein Assay is a widely used method for quantifying the amount of protein in a sample. It was developed by Oliver H. Lowry and colleagues in 1951 and has since become one of the most widely used protein assays due to its high sensitivity, accuracy, and reproducibility.
The lowry protein assay involves the reaction of proteins in the sample with a mixture of reagents, including copper ions and a Folin-Ciocalteu reagent. The resulting color change is then measured at a specific wavelength, allowing the protein concentration in the sample to be calculated.
The significance of the Lowry Protein Assay lies in its ability to accurately and reliably quantify protein concentrations in a wide variety of sample types, including biological fluids, tissues, and purified protein samples. It is used extensively in research, clinical, and industrial applications.
Some of the main applications of the Lowry Protein Assay include:
- Protein purification and characterization
- Enzyme kinetics studies
- Protein quantification in clinical samples
- Monitoring protein expression levels in cell culture
- Analysis of protein-protein interactions.
Materials
- Bovine serum albumin (BSA) or another protein standard
- Sample containing protein
- 2% (w/v) sodium carbonate
- 0.1% N sodium hydroxide
- 0.5% (w/v) copper sulfate
- 1% (v/v) Folin-Ciocalteu reagent
- Distilled water
- Microplate reader or spectrophotometer
Procedure
1 Prepare a protein standard curve
1) Prepare a series of dilutions of the BSA standard solution in distilled water, such as 0.5, 1, 2, 4, 6, and 8 µg/mL.
2) Add 2% (w/v) sodium carbonate to each standard and sample solution, and incubate at room temperature for 10 minutes to denature the proteins.
3) Add 0.5% (w/v) copper sulfate to each solution and incubate for 10 minutes at room temperature to form a copper-protein complex.
4) Add 1% (v/v) Folin-Ciocalteu reagent to each solution, mix well, and incubate for 30 minutes at room temperature.
5) Measure the absorbance of each solution at 750 nm using a microplate reader or spectrophotometer.
6) Plot the absorbance values of the standards against their respective protein concentrations and generate a standard curve.
2 Measure the protein concentration of the sample
1) Prepare the sample by diluting it with distilled water if necessary.
2) Add 2% (w/v) sodium carbonate to the sample and incubate at room temperature for 10 minutes to denature the proteins.
3) Add 0.5% (w/v) copper sulfate to the sample and incubate for 10 minutes at room temperature to form a copper-protein complex.
4) Add 1% (v/v) Folin-Ciocalteu reagent to the sample, mix well, and incubate for 30 minutes at room temperature.
5) Measure the absorbance of the sample at 750 nm using a microplate reader or spectrophotometer.
6) Determine the protein concentration of the sample using the standard curve.
Notes:
- This protocol assumes the use of a microplate reader or spectrophotometer with a 96-well plate.
- It is important to use the same type and amount of protein standard throughout the assay.
- Avoid using samples that contain reducing agents, detergents, or other interfering substances that may affect the assay accuracy.
- The assay can be adapted for use with other types of proteins or samples by optimizing the dilutions and incubation times.
I hope this protocol helps you in your protein quantification experiments. If you have any questions or need further assistance, please feel free to contact me.
