Technique and apparatus for automated fractionation of the contents of small centrifuge tubes: application to analytical ultracentrifugation.
An automated method is described for dividing the contents of small cylindrical centrifuge tubes into fractions deriving from laminae of solution as thin as 0.1 mm in the direction of the cylindrical axis. Experimental data are presented to demonstrate that fractions as small as 1 microliter may be prepared with a standard deviation of less than 3% in volume delivery and that negligible mixing occurs between the contents of adjacent fractions during the fractionation procedure. The method has been used to quantitate the gradients of a variety of radiolabeled proteins formed in sedimentation velocity and sedimentation equilibrium experiments. Sedimentation coefficients and molecular weights calculated from the gradients obtained agree well with literature values and with values obtained by optically scanning the centrifuge tubes (A.K. Attri and A.P. Minton, 1983, Anal. Biochem. 133, 142-152; 1984, Anal. Biochem. 136, 407-415). The present technique combines a spatial resolution equal to that of optical methods of gradient measurement with a sensitivity which may be several orders of magnitude greater, depending upon the specific activity of labeled solute.
D000818:Animals; D002417:Cattle; D002499:Centrifugation, Density Gradient; D005069:Evaluation Studies as Topic; D011506:Proteins / Q000302:isolation & purification*; D013053:Spectrophotometry; D014461:Ultracentrifugation / Q000295:instrumentation / Q000379:methods*
D011506:Proteins