Application scheme for determination of protein nucleic acid Content (ultraviolet absorption) -

Principle of measuring protein content

Because the benzene ring of tyrosine and tryptophan residues in protein molecules contains conjugated double bonds, the protein has the property of absorbing ultraviolet light, and the absorption peak is at 280nm wavelength. In this wavelength range, the light absorption value (A280) of protein solution is proportional to its content, which can be used for quantitative determination.

Principle of measuring nucleic acid content

Nucleic acids and their derivatives, nucleotides, nucleosides, purines and pyrimidines, have UV absorption properties, and their absorption peaks at 260nm wavelength. DNA concentration (ug/ml) =A260/0.024/L* dilution ratio (L is the thickness of the colorimetric tank 1cm)

Instruments and reagents

Macylab UL-5000 Nanodrop spectrophotometer

Standard protein solution. The content of bovine serum protein was determined by Kjeldahl method in advance, and 1mg/ml protein solution was prepared according to its purity.

Protein solution under test
Nucleic acid solution

operating steps

Drawing standard curve

Add various reagents to each test tube according to the table below and shake well. A quartz colorimetric cup with optical path of 1cm was selected to measure the 0A280 value of each tube solution at 280nm wavelength. The standard curve was drawn with the value of A280 as the ordinate and protein concentration as the abscise.

	1	2	3	4	5	6	7	8
Standard protein solution / ml	0	0.5	1.0	1.5	2.0	2.5	3.0	3.5
Distilled water / ml	4.0	3.5	3.0	2.5	2.0	1.5	1.0	0.5
Protein concentration / (mg/ml)	0	0.125	0.250	0.375	0.500	0.625	0.75	0.875
A280	0	0.105	0.156	0.221	0.320	0.373	0.482	0.542

Sample determination

Take 1ml of the protein solution to be measured, add 3ml of distilled water, shake well, measure the light absorption value at 280nm wavelength according to the above method, and find out the concentration of the protein to be measured from the standard curve.
Take 1ml of nucleic acid solution to be tested and add 3ml of distilled water to determine the concentration of nucleic acid.

Experimental result

The A value of the solution measured in the eight test tubes is shown in the figure

application scheme for determination of protein nucleic acid content (ultraviolet absorption) 1

The protein sample had A value of 0.063
The A value of the nucleic acid sample was 0.039

protein content

According to the equation, when A= 0.063, 0.0630 = 0.6123c + 0.0070.
C=0.0945mg/ml.
Because the solution is diluted four times, the protein concentration of the sample is
0.0945*4= 0.378mg /ml

Nucleic acid content

DNA concentration
= A260/0.024/L* dilution ratio
= 0.039/0.024 /1 * 4
= 7.8ug /ml

application scheme for determination of protein nucleic acid content (ultraviolet absorption) 2

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Application scheme for determination of protein nucleic acid Content (ultraviolet absorption)