3.2 Determine the sample's concentration Transfer 1.00 ml sample protein solution into a clean tube,add 3.00 ml distilled HO,then measure the mixed solution's As according to the above protoco 4.Results 4.1 Set the standard protein's concentration as X-axis,Azso as Y-axis,draw a standard curve. 4.2 Read the sample's concentration in the standard curve Notes: 1d280 varies greatly between different proteins (for a I mg/mL solution.from 0 up to 4 [for some tyrosine-rich wool proteins].although most values are in the range 0.5-1.5).It is best to measure absorb ces in th ge 0.05-1.0(between 10 and%of the incident radiation).At aroud 0.3 absorbanc (50%absorption).the accuracy is greatest 2.Bovine serum albumin is frequently used as a protein standard:I mg/mL has an A-8o of 0.66 3.If the solution is turbid.the apparent so will be increased by light scattering.Filtration (through a 0.2-um Millipore filter)or clarification of the solution by centrifugation can be oximate orretion can be applied by at this wave they contain particular chromophores)from the A280 4.At low concentrations,protein can be lost from solution by adsorption on the curve:the high ionic strength helps to prevent this.Inclusion of a nonionic detergent (0.01%Brij35)in the buffer may also help to prevent these losses. 卡i市t2而e il7 hromoph res (e.g.hen se If nucleic gives an accurate estimate of the protein content by removing the contribution to absorbance by nucleotides at 280 nm.by measuring the which is largely owing to the latter Protein (mg/mL)=1.554280-0.76 1260 protein in the posible prese of u ids the followi Protein(mg/mL)=(4235 42802.51 Protein(mg/mL)=0.183A230-0.075.8A260 6.Protein solutions obey Beer-Lambert's Law at 215 nm provided the absorbance is<2.0. 7.Strictly speaking.this value applies to the protein in6M guanidinium-HCl.but the value in buffer is generally within this value. nces in guanidinium-HCl 8.Sodium chloride,ammonium sulfate,borate,phosphate,and Tris do not interfere.whereas 0.1M acetate.succinate.citrate.phthalate.and barbiturate show high absorption at 215 nm. 9.The absorption of proteins in the range 215-225 nm is practically independent of pH between pH values4-8. has been determined.The average extinction coefficient for a I mg/mL solution of 40 serum proteins at 210 nm is 20.5 0.14.At this wavelength.a protein concentration of 2 ug/mL gives A=0.04