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Protein calculation using the Waddell method*
This methods assumes that you have an accurate spectrophotometer and that the buffer does not absorb in the far UV range.
Blank the spectrophotometer from 200 nm to 240 nm (if you have a scanning spectrophotometer) with a suitable blank and read the blank afterwards. Measure the absorbance of the peptide solution at 215 nm and 225 nm. The absorbance at 215 nm must be less than 0.5; if not, dilute with buffer and measure again. Measure the absorbance at both 215 nm and 225 nm. To determine the mg/ml: A215 – A225 * df * 0.144.
Divide the mg/ml by the MW of the peptide to determine the molar concentration and multiply this result by 1,000,000 to determine the μM concentration.
*W.J. Waddell J. Lab. Clin. Med, 48 (1956), pp. 311-314.