notified to come and inspect it. Failure to adhere to these instructions will void any insurance claims
we might seek and result in BioComp absorbing unnecessary expenses.
1.5 INSTRUMENT OVERVIEW
The Gradient Station combines the best of BioComp’s two premier gradient instruments in one.
The Gradient Master is embodied by a tilting and rotating disk on the right wing of the Piston Gradient
Fractionator.
Gradient forming is accomplished the the patented “tilted tube rotation” method invented by Dr.
David Coombs, the founder of BioComp. This remarkably simple and reproducible system involves
layering the 2 end point solutions in the tube, capping the tube and rotating it for short time at a
predetermined angle. The rotation of the wall of the tube smears the interface between the light and
heavy solutions, forming the desired gradient.
The Piston Gradient Fractionator (PGF) was also invented by Dr. Coombs (Coombs, 1974, Anal.
Biochem.
68:
95-1011) and offers a new dimension in fractionation. A piston is forced into the centrifuge
tube from above and it seals against the inside wall of the tube, forcing the gradient out of the tube
layer by layer. The size of the fraction taken is determined by the length of the piston stroke. A key part
of the evolution of the device has been in the shape of the piston face in contact with the gradient. It
converts horizontal bands of particles into vertical tubes traveling up the outlet tubing. The current
Trumpet Tip™ design doubles the peak height and halves the peak width compared with standard
needle puncture devices (see the web page for the actual data).
Dr. Coombs’ work with the purification of viruses led him to develop an exquisitely sensitive
band visualization system. T4 phage at >10
8
phage/ml can be visualized with the naked eye.
Ribosomal subunits are visible. This is possible for two reasons: light scattered by the particles from
the bright spot light shining underneath the tube is seen against the black background of the tube
holder. In addition, when the holder is filled with water before the tube is inserted, the outer surface of
the tube essentially disappears since the refractive index of the solution and the tube are nearly the
same. This lowers the background light scattering to such low levels that the faintest bands are visible.
In the future, we will provide UV sensitivity for particles that do not scatter visible light.
Another key to sensitive fractionation is the ability to clean out the tubing between fractions with
air or buffer. Often the top of a gradient contains large amounts of proteins, lipids or radioactivity that
smear into lower fractions if the tubing is not rinsed. This problem has been eliminated by injecting air
and/or buffer directly into the system at the point of gradient capture in the tip of the piston.
The software has been designed to be as simple as possible, but it will still take getting used to.
The piston’s downward movement at the start of the run is controlled by a dial. The further your turn
the dial, the faster the piston moves downward. As you manually bring the tip of the piston into the
tube, you will be asked to keep an eye on the outlet tubing to spot the
first drop
to leave the tubing.
This will become the “TOP” of every gradient, and fractionation can proceed accurately below this
point if you press the RESET key to zero the device at this position. The rest is described below.
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