Quantitation of Protein:Ultraviolet Absorption Method

【 Purpose 】
1.Master the principle method of ultraviolet absorption method to quantitate protein.
2.Learn the operational and application method of ultraviolet spectrophotometer.
【 Principle 】
There are amino acid residues with aromatic ring in protein molecule (such as tyrosine and tryptophan), so there is maximal ultraviolet absorption at 280nm, at which the relationship between absorbance and protein concentration is a straight line in a definite range. So we can determine protein concentration by ultraviolet absorption method.
Ultraviolet absorption method can determine the protein concentration range from 0.1 to 0.5 mg/ml. This method is easy to perform. It determines protein concentration promptly, does not consume sample and cannot be interfered by low salt concentration. So this method is widely used in the preparation of protein.
【 Apparatus 】
Test tubes and test tube shelf, 50ml flasks, Pipets, Ultraviolet spectrophotometer
【 Reagents 】
1. Standard protein solution: Exactly make casein solution of 2mg/ml.
2. Sample solution: Make 0.5mg/ml casein solution as unknown sample solution.
【 Procedures 】
1. Draw calibration curve
Number eight test tubes, and add reagents as the following table

	0	1	2	3	4	5	6
Standard protein concentration(ml) Distilled water(ml) Protein concentration(mg/ml)	0   8.0 0	0.5   7.5 0.125	1   7.0 0.250	1.5   6.5 0.375	2.0   6.0 0.500	2.5   5.5 0.625	3.0   5.0 0.750

Mix up after adding, measure absorbance at 280nm by 751 - ultraviolet spectrophotometer, while the test tube numbered 0 is contrast. Draw absorbance - protein concentration curve, while the absorbance is y-axis, the standard protein concentration is x-axis.
2. Sample assay
Imbibe 4.0ml of sample solution, add 4.0ml of distilled water, measure ab­sorbance at 280nm.
【 Results 】
According to the absorbance of sample solution, check out the protein concentration in the sample solution from the calibration curve.