I purified smURFP was concentrated to be ~3.5 mL. and measured absorption spectrum of the sample by diluting 100-times in a buffer. Absorbance at 280 nm of the diluted sample was 0.132 for finding the concentration of the sample do I have to multiply 100 to the value obtained to get the real concentration value ?
And also, how do I find the wight of the protein ?
Yes, you should multiply the absorbance by the dilution factor 100. This gives you the A280 of the original solution. To get the protein concentration, you need to know the extinction coefficient of the protein. There are on-line tools for calculating the extinction coefficient of proteins based on their amino acid compositions, but they require that the measurement be made on fully denatured protein. The absorbance measurement should be done in 6M guanidine HCl to ensure that the protein is fully denatured.
Alternately, you could use one of the commercially available protein assay reagents, such as Bradford, BCA, or Lowry, and a standard protein such as bovine serum albumin with which to prepare a standard curve.
