The ultraviolet hydrolight method measures protein concentration.

. Objective: To master
principle of determining the content of
proteine
by ultraviolet hydrolights, and to be familiar with the use of ultraviolet
hydrolight meters
technology.:protein molecules contain conjugated double-bonds tyrosine, tryptophan and other aromatic
amino acids
. They have the nature of absorbing ultraviolet light, its absorption peak at 280nm wavelength, and in this wavelength absorption peak light density value is directly related to its concentration, so it can be used as the basis for quantitative determination of proteins, but because of the different content of various proteins tyrosine and tryptophan, it is necessary to accurately quantify, it is necessary to test the protein's pure products as a standard to compare, or already know its de-light coefficient as a reference. In addition, many impurities at 280nm wavelength also have - fixed absorption capacity, may occur interference. In particular,
effects of
nucleic acids (pyridine and steroids) are more severe. However, the maximum absorption peak of nucleic acids is 260nm. Therefore, when nucleic acids are present in the solution at the same time, OD
260
nm and OD
280
nm. Then, according to the ratio of absorption of the two wavelengths, the true content of the protein is calculated by empirical formula correction to eliminate the effects of nucleic acids.
method is simple and fast, and does not consume samples (can be recycled), and is used for micro-measurement of purified proteins. Main disadvantages: When the protein to be tested and standard protein tyrosine and tryptophan content difference is large, then a -fixed error, mixed with nucleic acids must be determined 280nm and 260nm two OD values, and then according to the formula to calculate the protein content.take
test tubes
7 units, operate according to the table:tube mix well, in the quartz cup, with ultraviolet trachea photomeometer, with blank tube to adjust zero points, respectively, at 28 The light density of each tube is measured at 0nm and 260nm wavelengths
(calculation)
(i) the sample protein content is calculated directly according to the optical density value (OD280nm) of the standard liquid and the liquid to be measured, or from the standard curve.
the standard tube light density value as the ordinate, the protein concentration as the horizontal coordinates to draw the standard curve, and then according to the measurement of the tube light density value, directly check the standard curve to find the protein content in the sample.
1. Select a protein pure product similar to the amino acid composition of the protein in the sample under test, diluted with physiological saline to a concentration of lmg/m1, as a dilution standard protein solution.
2. Dilute the sample protein to the concentration of about lmg/m1 with physiological saline, i.e. dilute the sample protein solution.
(ii) Using the empirical formula to directly calculate the protein content of the sample,
accurately absorbed
serum
sample 0.1 ml, placed in 50 ml
capacity bottle
, diluted with physiological saline to the scale, that is, 500 times diluted, at 280nm and 260nm wavelengths measured light density values, and then calculated according to the following formula.
1, OD
280
/0D
260
<1.5, using lowry-Kalokar formula: protein content of
samples (mg/m1) - 1.45OD
280
0.74OD2
60 2, OD
280
/OD
260
>1.5, Lamber-Beer's Law Calculated:
protein content (mg/m1) - OD
280
/K×L (OD
280
/6.3×1) ×10g// L
samples: Bovine serum albumin E
1%
cm=6.3 (100ml/cm.g)
K: g molecule
1%
; cm: percentage absorbance absorption coefficient;
Note: There are differences in tyrosine and tryptophan content in different proteins, so the protein amino acid composition of the standard tube and the measuring tube should be similar to reduce the error.
(i)
UV-9200 UV-9200 UV estrometer
(ii) 50 ml capacity bottle

reagents

) (i) Physiological saline
(ii) clear protein (human or cattle) pure crystal
(iii) sample protein to be tested: samples of proteins measured by double shrinking are diluted with physiological saline. .