Boston Biomedical Research Institute
Analytical Ultracentrifugation Research Laboratory
Walter F. Stafford, Ph.D., Director (retired)

Sample Preparation for Analytical Ultracentrifugation:

For initial runs, sample concentrations should be from 0.3 to 1.0 mg/ml for use with the Rayleigh optical system (refractive index) or in the range of 0.4 to 1.0 a.u. at the appropriate wavelength for the absorbance optics.


Preferably, samples should be of the highest purity possible with gel filtration being the last step to remove aggregates. Remember: "Garbage in - garbage out"

Samples also should be at "osmotic" equilibrium with their respective buffers This can be achieved either by 24 hour dialysis or by gel filtration. Either ordinary column gel filtration by sephadex, FPLC or HPLC can be used, or "spin" columns can be used (e.g. cf. Christopherson and Jones, Anal. Biochem 100, 184-187 (1979)). Spin columns are convenient because they are fast and there is effectively no dilution during buffer exchange.

For samples prepared by dialysis, it is best to ship the samples still in their dialysis bags in about 25-50ml of dialysis buffer. Samples seem to survive shipping better if they are shipped in a dialysis bag still dialyzing. Try to squeeze all the air out of the bag to avoid surface denaturation at the air-solution interface. More importantly, the extra time under dialysis will ensure that the sample will be well enough equilibrated that the dilaysate can be used as an optical blank without have to re-dialyze. Remember we are subtracting two very large numbers (the refractive index of the dialysate from the refractive index of the buffer that the sample is in) to get the refractive index contribution of the protein. So, an exhaustive dialysis is required against the last buffer change.

Samples that are prepared by ordinary gel filtration or spin columns, must accompanied by about 25-50 ml of the column buffer which will be used to make dilutions and as an optical reference. This aliquot of buffer must be the identical buffer that was used to equilibrate the column. Since the refractive index match between the sample buffer and the reference buffer must be exact, an aliquot of buffer of nominally the same composition will not match sufficiently well to act as reference.

Samples can be shipped for overnight express delivery on wet ice. Ice and sample tubes should be wrapped in a knotted plastic bag which is in turn placed inside another knotted plastic bag and then placed in an appropriate insulated shipping container. This system of double bagging works well and has resulted in very few leaking packages. Double-bagged "Zip-Lock" bags also work well.