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proteinconcentration determination - UV absorption
"principle"
because of the existence of tyrosine and tryptophan with conjugated double bonds in the protein, Therefore, the protein solution has a UV absorption peak at 280nm. Within a certain concentration range, the absorbentity of the protein solution at this wavelength is directly related to its concentration, so this property can be used for protein quantification.
is a rapid, simple, non-consuming sample, low-concentration salt without interference determination, can be determined 0.1 to 1.0mg/mL protein solution.
because the protein's UV absorption peak often changes due to pH changes, so the application of this method should pay attention to the pH of the solution. The pH of the best sample is consistent with the pH of the standard curve.
This method has a large protein error in determining the content of tyrosine and tryptophan in standard proteins, so it is applicable to the determination of samples with similar content ofamino acids such as standard protein tyrosine and tryptophan, and if the sample contains UV-absorbing substances such as radon and zirconium.
e.g. mixed withnucleic acid, which absorbs ultraviolet light at a wavelength of 280nm, but absorbs more ultraviolet light from 260nm. In contrast, the UV absorption value of 280nm is greater than the UV absorption value of 260nm. The interference between nucleic acids and protein concentration determination can be corrected by using 280/260 absorption difference method.
"methods and steps"
1, production of standard curves
feed 7"> test tubes", numbered and then added to the standard protein solution and sodium hydroxide solution in turn.
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1
2
3
4
5>5>5
6
standard protein solution/mL
0
0.5
1.0
1.5
2.0
2.5
0.1mol/L NaOH/mL
4.0
3.5
3.0
2.5
2.0
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gabi, stir well, choose quartzlight-thromometer to measure A280.
< p style is "text-align:left;" > is a horizontal coordinate of the number of milligrams per tube of standard protein, and the corresponding A280 value is the ordinate, making a standard graph.2, protein sample determination
< "text-align:left;" >(1) standard curve methodexperiment samples can be diluted with homemade casein solution in milk plus 0.1mol/LH.
< p style" text-align: left;" > put the sample protein solution into a quartz cuvette, measured A280, and measured the protein concentration against the standard curve.n - the number of casein milligrams detected from the standard curve.
(2) 280nm and 260nm absorption differential method:
to determine the absorption value of protein samples at 280nm and 260nm respectively, according to the following formula to calculate the protein mass concentration.
protein mass concentration (mg/mL) - 1.45 A280 - 0.74 A260
< A280 - the absorbance measured by the protein solution at 280nm;
A260 - the absorbance measured by the protein solution at 260nm.
< p style is "text-align: left;" > the light absorption rate of different proteins and nucleic acids is not exactly the same, and errors may still be generated in this way..
