Results and analysis:

1. 16. Switch ON the UV/Visible spectrophotometer and allow it 30 minutes warm up.

17. Set the instrument to absorbance mode and wavelength to 280 nm (Note 3).

18. Zero the reading taking first fraction as a blank.

19. Measure the absorbance of all the fractions (washing of cuvette in-between is not required for any of the fractions).

20. Record the absorbance in an observation table (Table 23.1).

 

Table 23.1: Table for recording absorbance of collected fractions

1. 21. Plot A₂₈₀ against elution volume to obtain the chromatogram of standard proteins. The chromatogram should give six distinct peaks; label the peaks from 1 – 6 with increasing elution time.

22. Equilibrate the column with two bed volumes of buffer and repeat the procedure for the given protein of unknown molecular weight.

23. Plot A₂₈₀ of the unknown protein fractions against elution volume to obtain its chromatogram (Note 4).

24. Calculate the elution volumes of the reference proteins as well as the unknown protein.

25. Using elution volumes, V₀ , and V_t , calculate the K_av of the proteins (Table 23.2):

(23.1)

1. 26. Calculate the log₁₀ (Mol. wt.) of the standard proteins (Table 23.2).