Rapid and accurate microfractionation of the contents of small centrifuge tubes: application in the measurement of molecular weight of proteins via sedimentation equilibrium.
The contents of small centrifuge tubes containing solutions of radiolabeled proteins that had been centrifuged to sedimentation equilibrium were fractionated using a new device based upon the mechanical design of Attri and Minton (Anal. Biochem. 152, 319-328, 1986). Individual fractions, corresponding to laminae of 0.15 mm column height within the tube, were collected using one of two methods: (a) automatic mixing with scintillation fluid and delivery to vials mounted in a fraction collector, or (b) collection of undiluted fractions on scintillation vial caps impregnated with solid scintillator. Gradients of protein concentration were obtained via scintillation counting of sequential fractions. Molecular weights of proteins ranging from 4 x 10(4) to 3.5 x 10(5) were calculated by fitting the theoretical expression for sedimentation equilibrium of an ideal homogeneous solute to the experimental gradients. The values so obtained agree well with values obtained by optical scanning of the unfractionated centrifuge tube, and with values obtained from the literature.
Darawshe
S
S
Laboratory of Biochemical Pharmacology, National Institute of Diabetes, Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892.
Rivas
G
G
Minton
A P
AP
D000818:Animals; D002499:Centrifugation, Density Gradient / Q000379:methods; D005591:Chemical Fractionation / Q000379:methods; D002645:Chickens; D006801:Humans; D007700:Kinetics; D008832:Microchemistry / Q000379:methods; D008970:Molecular Weight; D011506:Proteins / Q000032:analysis* / Q000737:chemistry; D012588:Scintillation Counting; D014316:Tritium
D011506:Proteins; D014316:Tritium