Description of the Instrumentation

SEC-LS system

The size exclusion chromatography is carried out on one of the following columns:

  • Superose 6 HR10/30 column (Pharmacia), which separates proteins with molecular weights that range from approximately 30 kDa to 2x106 Da.
  • Superdex 200 HR10/30 column (Pharmacia), which separates proteins with molecular weights that range from approximately 10 to 600 kDa.
  • Superdex 75 HR10/30 column (Pharmacia), which separates proteins with molecular weights that range from approximately 1 to 70 kDa.
  • Superdex peptide HR10/30 column (Pharmacia), which separates proteins with molecular weights that range from approximately 100 to 5000 Da.

The total column volume is ~24 ml and the standard buffer is 20 mM HEPES, 150 mM NaCl, 1 mM EDTA with/without 1 mM DTT, pH 7.5. The SEC is carried out at a flow rate from 0.3 ml/min to 0.6  ml/min (depending on the column used) at room temperature and the run time is about 1 hour. Although standard SEC runs are carried out in an "analytical mode" (without collection of the eluting peaks), fractions can be collected upon request and at an additional charge. Because of the extended equilibration time that is needed to establish a stable LS baseline and the additional standards that need to be analyzed after changing buffers, we encourage use of the standard HEPES buffer. Samples that must be subjected to SEC in other buffers will be subject to an additional charge and longer turn-around. The SEC column is connected on-line with the following HPLC flow detectors:

  • Multiwavelength UV/VIS detector
    (Waters 996 Photodiode Array-PDA detector, Waters, Milford, MA)

  • LS detector
    (DAWN®- EOS, Wyatt Technology, Santa Barbara, CA)
  • RI detector
    (OPTILAB rEX, Wyatt Technology, Santa Barbara, CA)

The system is calibrated using three to five proteins that extend from 1 to 475 kDa. The MW is determined also from the absolute measurement of the scattered light intensity measured at 18 different angles. Although the UV signal does not enter directly into the MW calculation, comparison of the UV/RI signal ratio across the peaks of interest provide information relating to the homogeneity of the sample. Thus, if there are several peaks that arise from different oligomers of the same polypeptide, the UV/RI ratio should remain unchanged for all these peaks throughout the chromatogram. This value can be used also to independently estimate the extinction coefficient for the protein.