1- Making Density GradientsPre-formed discontinuous gradientsPre-formed continuous gradientsSelf-generated gradients
2- Density Gradient Harvesting
Training File 2 topics
Discontinuous gradient by overlayering
If we make up, for example 3 aqueous solutions of, for example, iodixanol (10%, 20% and 30%, w/v) we can make a discontinuous gradient by overlayering; each solution (starting with the densest) is overlayered with successively lighter solutions from either a pipette or syringe attached to a wide-bore flat-tipped metal tube (cannula). Overlayering is best accomplished if the tube is tilted (as in C) and the tip of the pipette or syringe placed approx 0.5 cm above the meniscus.
Discontinuous gradient by underlayering
The less commonly used method of underlayering (low density end first) is in fact the more satisfactory method. It is only easily accomplished using a syringe and metal cannula and the hemispherical section of the bottom of the tube aids smooth flow.After adding the least dense solution, the next most dense solution is taken into the syringe if for example you need to layer 3 ml, then take up approx 5 ml and expel the liquid to the 4 ml mark (this ensures there are no air bubbles in the cannula. Wipe the outside of the cannula with a tissue to remove any excess liquid and introduce the tip of the cannula to the bottom of the tube, moving it smoothly down the wall of the tube. Smoothly introduce 3 ml of the solution. Depressing the syringe plunger to the 1ml mark rather than to the bottom of the barrel is both more accurate and ensures any air bubbles, trapped on the plunger, do not enter the tube. After a few seconds withdraw the syringe, again keeping the tip of the cannula against the wall of the tube and repeat the procedure.
Diffusion of a discontinuous gradient
If a discontinuous gradient of say 3 ml each of 10%, 20%, 30% and 40% iodixanol in a 14 ml tube is allowed to stand for several hours at room temperature or overnight at 4C, then the sharp discontinuities of the stepped density profile become smoothed out by diffusion of the solute across each interface and eventually the gradient will become smooth and continuous. This is a commonly used strategy to make a continuous gradient.A linear gradient will be produced if the density increment between each step is the same and the volume of each step is the same. If either the volume of one or more of the steps is changed or the density interval of each step is changed the shape of the gradient can be made concave or convex or irregular in order to accentuate the separation of particular particles.If the diffusion time and temperature are well controlled the gradient shapes are highly reproducible.
Rapid formation of continuous from discontinuous gradient
If the tube containing the discontinuous gradient is capped and very carefully and smoothly rotated to a horizontal position, the interfacial area increases and the distance between each interface decreases, so diffusion and formation of a continuous gradient will occur much more rapidly. At room temperature, a continuous gradient of iodixanol in a 14 ml tube can be formed in approx 45 min. This should not be attempted the morning after the night before.
Two-chamber device for continuous gradients
Two identical chambers (A and B) connected by a tapped channel (T) contain equal volumes of the high density and low density solutions respectively and identical magnetic stirring bars (SB). Chamber B, which sits on a magnetic stirrer (M), bears a delivery tube which reaches to the bottom of a centrifuge tube via a low-pulsation peristaltic pump (P). When the levels of liquid in the two chambers fall synchronously, dense solution from A mixes continually with the lighter solution in B so that the pump delivers a solution of ever-increasing density to the bottom of the centrifuge tube. The two stirring bars ensure that the level of liquid in A and B is the same.If the two solutions have very different densities and if the stirring is not sufficiently vigorous, the dense solution may flow under the lighter solution. If the placement of the two solutions is reversed, then the tendency of the less dense solution two float up through the denser solution in B will improve mixing considerably. In this format the tip of the delivery tube needs to be placed against the wall of the tube, near its top, so that solution of decreasing density flows to the bottom. If the flow down the wall of the tube is not continuous (but in the form of drops) this can also cause mixing. Use of the Labconco Auto Densi-flow machine (see Graphic 31) to deliver the gradient into the tube avoids this problem totally in the dense-end first mode.A single two-chamber gradient maker can be used to create multiple gradients but any manifold in the delivery line must be situated before P.
The Gradient Master
The Gradient Master (and its less expensive small brother, the Gradient Mate) are manufactured by Biocomp (Fredericton, New Brunswick, Canada). The two solutions (e.g. 10% and 40% iodixanol) are layered in a tube which is capped and placed in the vertical barrel (B) which accommodates up to 6 tubes (depending on the tube volume). The barrel then tilts automatically (usually to 80 to the vertical) and rotates at approx 20 rpm for 2-3 min. The tilting increases the interfacial area between the two solutions and the gradient is formed by the controlled mixing. The barrel returns to a vertical position at the end of the mixing period. The viscosity of the two solutions and the shape of the desired gradient influence the required values for the time and rpm parameters.It is a particularly good device to use if the sample is to be included in one or both of the layers (see Training File 1 for sample/gradient handling formats).
Gradient Master profiles from 10% and 40% iodixanol at 80 and 20 rpm: effect of time
Increasing the time of rotation generally makes the gradient more linear and more shallow
There are two traditional types of rotor: swinging-bucket and fixed-angle. The swinging-bucket rotor is by far the most widely used for density gradient centrifugation.In the swinging-bucket rotor, at rest, the tube and bucket are vertical and the meniscus of the liquid is at 90 to the earths vertical centrifugal field. During acceleration of the rotor the bucket, tube and meniscus reorient through 90 in the spinning rotors radial centrifugal field. Any gradient in the tube reorients with the tube so that interfaces are also always perpendicular to the centrifugal field.
Fixed-angle and vertical rotors
The other two types of rotor are fixed-angle and vertical.A fixed angle rotor is normally used for simple pelleting of particles but there are examples in which these rotors are used for gradient work.A vertical rotor is the most efficient way of doing density gradient separations of membranes, viruses and macromolecules (see next 11 and 12).The tube is maintained in vertical position and the gradient reorients in the tube, illustrated with a discontinuous gradient in the graphic. A gradient will reorient in the same way in a fixed-angle rotor tube. The acceleration and deceleration between 0 and 2000 rpm, during which the reorientation occurs must be achieved slowly and smoothly to avoid disturbance to the gradient.
Sedimentation path length of rotors
In a swinging-bucket rotor the sedimentation path length is the length of the tube.In a vertical rotor the sedimentation path length is the diameter of the tube.The vertical rotor is therefore the most efficient rotor; at the same RCF, particles will reach their banding density much more quickly in a vertical rotor than in a swinging-bucket rotor. The fixed-angle rotor would occupy an intermediate position. But in a vertical rotor sedimenting particles cannot encounter the wall of the tube in the same way as they do in a fixed-angle rotor.In addition, for tubes of the same volume and dimensions (rotating at the same rpm), the hydrostatic pressure experienced by a particle is much less in a vertical rotor than in a swinging-bucket rotor, since the height of the liquid column is much smaller. The hydrostatic pressure is a function of the square of the height of the liquid column and this pressure has been shown to damage some organelles.In short, for density gradient centrifugation, the vertical rotor has none of the disadvantages of either the swinging-bucket rotor or the fixed-angle rotor.
Sample/gradient in vertical rotor
The radial thickness of any sample placed on top of a density gradient (A) will also be much smaller in a spinning vertical rotor (B) than in a swinging bucket rotor of the same tube volume. In any separation based on sedimentation velocity, the resolution of the zones, which is dependent on the radial thickness of the sample, may potentially be much better in a vertical rotor than in a swinging-bucket rotor. This factor may outweigh the possible disadvantage of the shorter path length of the vertical rotor.The potential for sample/gradient interfacial instability is also less in a vertical rotor.
Self-generated gradients At 360,000gav iodixanol molecules will start to sediment
Some gradient solutes, whose molecules are sufficiently dense, will sediment, just as any other particle does, if placed in a sufficiently high centrifugal field. As a result of the sedimentation of the molecules, a solute gradient is formed this is called a self-generated gradient. This phenomenon is well established with heavy metal salts such as CsCl and Cs2SO4 which are used for the banding of DNA and RNA. But some of the newer gradient solutes such as Nycodenz and iodixanol will do this as well. Becau