The QUANTITATIVE DETERMINATION of PROTEINS
The QUANTITATIVE DETERMINATION of PROTEINS
AIM : Protein quantitation enables us to find unknown concentration via making a comparison with known protein standards.
INTRODUCTION: Proteins are large, complex molecules that play many critical roles in the body.(1) Most genes contain the information needed to make functional molecules called proteins. (A few genes produce other molecules that help the cell assemble proteins.) (2) Hemoglobin is a protein in your red blood cells that carries oxygen to your body's organs and tissues and transports carbon dioxide from your organs and tissues back to your lungs.(3)
UV absorption: Sensitivity of this method is moderate. It can detect proteins in the range of 50-100 µg. In this method, no reagents are required, the liquid protein sample is monitored under UV absorption at OD 280nm.
Lowry assay: This is a highly sensitive and gives accurate results. It detects proteins at low concentrations of 2-5 µg
Bradford assay: This is a very sensitive method and simple dye binding assay. This method uses Coomassie brilliant blue-250 dye that binds with negatively charged protein molecules.
Biuret method: Sensitivity of this method is very low. It requires high protein levels from 1-20 mg of protein. The reagent used in this method are Copper sulfate, Sodium hydroxide, and Potassium Sodium Tartrate. (4)
The Beer-Lambert law is a linear relationship between the absorbance and the concentration, molar absorption coefficient and optical coefficient of a solution: (5)
MATERIALS : - BSA (1 mg /ml) solution - Bradford reagent 1X dye solution
- Eppendorf 1.5 ml tubes - 96 – well plate
METHOD : The standard protocol can be performed in a 250 μl microplate assay. The linear range of these
assays for BSA is 125-1,000 μg/ml.
2. Remove the 1X dye reagent from 4oC storage and let it warm to ambient temperature. Invert the 1X dye reagent a few times before use.
3. Pipette each standard sample solution into separate clean test tubes as shown in the sample table. Add the 1X dye reagent to each tube and invert.
4. Incubate at room temperature for at least 5 min. Samples should NOT be incubated longer than 1 hour at room temperature.
5. Set the spectrophotometer to 595 nm. Measure the absorbance of the standards including Blank.
RESULT :
0.907 = 0.9316x – 0.2628 1.1698 = 0.9316x X = 1.255
1.255 µg in 100 µl unknown protein solution:
1.255 µg / 1000 µl = 0.00125 µg / µl protein concentation of the unknown tube.
DISCUSSION : Apart from proteins, other substances can also interact with and absorb light. This situation negatively affects the correct result of the experiment. Therefore, the quantitation of products with high purity is almost correct.
REFERENCES : (1) https://medlineplus.gov/genetics/understanding/howgeneswork/protein/
(2) https://medlineplus.gov/genetics/understanding/howgeneswork/makingprotein/
(3) https://www.mayoclinic.org/tests-procedures/hemoglobin-test/about/pac-20385075
(4) https://info.gbiosciences.com/blog/protein-estimation-methods
(5) https://www.edinst.com/us/blog/the-beer-lambert-law/
