Protocol for accurate determination of concentration of pure protein

Main steps:

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1. Place 1ml of 6M GuHCl, 20 mM NaP, pH 6.5 into a quartz cuvette and zero it at 280 nm on a spectrophotometer.
2. Add 10-50 μl of protein solution containing about 0.1- 0.3 mg of the target protein into the same cuvette, seal with parafilm and mix by tipping upside down. For a reliable result the reading should be between 0.1 and 1.
3. 3Calculate concentration of protein using Abs 0.1% (1mg/ml) (this is given in the ProtParam data in Expasy data base for your protein). Multiply by dilution factor.
4. Using the same protein solution make a calibration plot for Bradford assay and calculate accurate factor which you can then use for accurate determination of concentration of this protein by Bradford method.
5. Place 1ml of appropriate buffer (for example buffer A, PBS or buffer A+0.1M NaCl) into a quartz cuvette and zero it in the range 240-340nm. Using the same protein solution, add the same volume as in step 2 and take spectrum from 240 to 340nm. If there is no significant light scattering between 300 and 340nm, calculate the extinction coefficient (at 280nm or at maximum) for the target protein under non-denaturing conditions (in the given buffer).