An Experiment Using Sucrose Density Gradients in the Undergraduate Biochemistry Laboratory Sandra L. Turchi and Monica Weiss Millersville University, Millersville. PA 17551 Macromolecules as well as subrellular components can he separated on the basis of theirrnolerular weight and volume b$ density gradient centrifugation. sucrose-density gradients are usually made either by using a density gradient maker or by carefully layering, into a centrifuge tube, solutions of sucrose varying in concentrations from 60% to 30% (11. . ,~ During centrifugation, a molecule will migrate to a zone in the tube where the densitv of the sucrose solution is eoual to the density of the rno1ec;le. Stable hands of the indikdual components will he formed in the gradient (2).This phenomenon is the basis of the isopycnic or equal density centrifugation (3). ~ r a d i e n centrifugation t experiments are usually reserved foreraduate courses and research due to the type . . of sophisticated and expensive equipment needed, such as an ulrracentrifuae. This experiment is one that ran be performed in an undergraduate "biochemistry laboratory. 1t is based on a model system for gradient centrifugation designed by L. Mullenger (4). I t employs a simple bench top centrifuge, a freezer, and a relatively simple method to prepare sucrose density gradients. The sucrose gradient solutions are frozen during preparation. Experimental
Centrifugation To carry out the isopycnic centrifugation, layer the prepared cellular mixture (5 mL) onto one of the thawed gradients, being careful not to disturb the gradient. Centrifuge at 6500 rpm for 30 min. Remove the tubes so as not to disturb the layers. Clamp in a vertical position. Note where the intense blue layers form. Fractionate the gradient by removing 5 mL at a time with a pipet. If this is done gently, the zone below will not be disturbed. Determine the absorbance at 550 nm. Plot absorbance vs. density. The second eradient is used to determine the linearitv of the gradient. ~raciionateas above. Weigh each fraction in a dry preweighed beaker. Convert to density. Plot density vs. fraction number. Discussion The experiment described above is simple and inexpensive to oerform. The seoaration of bacteria and veast cells is easily iisualized by ~ h e . ~ u r hands ~ l e of crystal \ h e r , which stains the cells. Fractionation of the sucrose gradient is easily done since the layers appear as shades of violet correspondinn to the different density zones. Furthermore, the u k x e n t r i f u g e is replaced by a bench top centrifuge. ~ k n s i ty gradients can he prepared in a number of different ways. The method explained above is based on the principle of freeze fractionation and is accurate and reproducible.
Gradient Preparation Gradients are prepared in thin-wall polyallomer tubes. Each tube is filled with 40 mL of 25%sucrose solution and placed upright in a freezer.The day before the gradients are to be used, they are placed in a refrigerator to thaw. It is important that the tubes remain upright and not be disturbed.A continuousgradient from about 15% (top) to about 45% (bottom) will form. Each student should have two gradients (5).
Acknowledgment We wish to thank the Millersville University Alumni Association for their financial support in the form of a NeimeyerHodgson Research Grant. We also want to express our appreciation to Jennifer Fisher for her expert secretarial services.
Biological Sample Preparation
Literature Cited
Prepare nutrient broth cultures of Saccharomycescereuisiaeand Escherichia coli (6).Any nonpathogenic procaryote can be substituted for E. coli. Incubate both flasks for 24 h a t 37 OC. Prior to the laboratory session, add a solution of crystal violet (1 mL, 0.02%wlv) to each flask. Let these stand for 1 h, then centrifuge each culture and wash the pellets with saline (0.9%) until clear. Suspend each pellet in phosphate buffer (40 mL, pH 7.0, 50 mM). Mix final suspensions together and refrigerate until needed.
1. Cmper. T.G.Tho Toola ofBiochamistry; Wilcy New York, 1977: pp 326347. 2. Clark, J.M;Suitzer,R.L.E~perimontolBioehemiafry; W.H.Freemsn:SanFrancisco, 1977: p p 37-62. 3. Bohinsky, R. C. Modern Concepta in Bioehemiatry;Allyn sad Bacon: Boston,1983: p p
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