Several methods of centrifuge separation.

methods of centrifugal separation, preparation type ultra-high speed
centrifuge
several separation methods:
A. Differential centrifugation:
centrifugal force is increased one by one, each time some of the parts in the sample solution can be sethered. Differential centrifuge is one of the most commonly used methods. In this
, the
tube is initially filled with an equal sample solution. By centrifugation at a certain speed for a certain period of time, two parts can be obtained: precipitation and semen. Most unwanted large particles are usually dropped at the first centrifuge. At this point, most of the required parts remain in the upper liquid. The collected upper liquid is then centrifuged at a higher speed, depositing the desired particles. The time of centrifugation should be chosen correctly so that most of the smaller particles that are not needed remain in the upper liquid. Further centrifugation of the resulting precipitation and semen can be carried out until the desired separation purity is achieved. Differential centrifugation is characterized by simple operation, but low separation purity.
B. Density gradient centrifugation:
separate several or all of the parts in the sample at the same time, with good resolution.
(1) rate zone band method (rate zonal): depending on the size of the different particles in the sample and the seation velocity (S). The approximate steps are as follows: a density gradient solution is loaded into the centrifuge tube, and the density of the solution gradually increases from the top to the bottom of the centrifuge tube (positive gradient). Carefully add the samples that need to be separated to the top of the density gradient solution. The sample forms a negative gradient on the surface of the gradient solution. Because particles of different sizes move at different speeds in the gradient under the action of centrifugal forces, several separate sample zones are formed after centrifugation. Note: The density of the sample particles must be greater than the density of any point in the gradient fluid injection. The centrifugation process must stop before the zone strap reaches the bottom of the tube.
(2) iso-density centrifugation method (isopycnic): separate according to the different densities of particles. During centrifuges, particles move to the same density as itself to form zones. The density sample is selected so that the range of gradients includes the density of all particles to be separated. Samples can be distributed on top of density gradient particles or evenly in density gradients. After centrifugal, the sample particles reach their equilibrium point. Note: The separation of particles after equilibrium is entirely determined by their density, independent of time, at which point the centrifugal speed can only be changed to the relative position of the zone.
2. Density gradient analysis method 1) gradient medium properties and choices: A, should have the nature: the principle of selection of gradient substances is to meet the basic requirements of the separation method, an ideal density material standard it should be: - the resulting solution density should include the desired density range. Has certain properties, such as refractive index, according to which its concentration can be determined. The resulting solution has a low viscosity. Do not damage the isolated sample. Easy removal after centrifuge separation. The analysis of separation integrals is not hindered. B, common media types: Table 1, commonly used gradient materials at a density of 20 degrees C B. Gradient media application range: Table 2, iso-density gradient medium application is very good, good can -- not applicable to Table 3, a variety of large molecules in the sucrose gradient liquid approximate density (2), gradient solution preparation: calculation dilution (chapter III, part 1)
(3), gradient shape shape: line type, isosonic type, step type, flat type, steep gradient index. Gradient shapes are important for the success of separation: the most commonly used are line gradients, which are suitable for separating
proteins
, enzymes,
hormones
, ucose glycosome subbases, and some plant viruses; Isomehic gradients and long columns enhance separation capabilities and are suitable for separating kerucleosome sub-base, poly-ucose and plant viruses. B. Gradient column preparation: Gradient column can be prepared by hand or gradient semi-injection method: to reduce centrifugal time, or separate samples less available half-injection method: half-pipe paved gradient medium, intermediate sample, top with Buffer or liquid stone oil.
(4), dosing method and dosing: add the sample to the gradient column, needle tip and centrifuge tube into a 45-60 degree angle, slowly flow the sample along the pipe wall to the liquid surface, for
. DNA
a class of fragile samples that are easily broken, and needles should be replaced with pipet tubes with large apertures to avoid shear force on the cutting of the sample. The sample concentration is 1/10 (W/W) of the minimum density of the gradient column.
(5), roor selection and effect
(6), separation zone belt recovery and detection of centrifugal area sample recovery methods are basically four:
a. puncture method puncture centrifugal tube bottom, so that the gradient solution drip, a cap with a suitable valve on the top of the centrifuge tube, can control the drip speed.
b. Siphon method: a capillary tube gently inserted into the bottom of the tube, as far as possible to prevent gradient jitter, with a trace pump gradually drip, to a certain number of drops or volume part of the charge.
c. pressurization method: through a needle tube to pump high-density liquid into the bottom of the gradient centrifuge tube, partially collected out of the solution.
d. cutting method: using a special cutting knife to cut the desired area belt. Zone band detection: The so-called zone band detection, which is actually the monitoring of the separation material in the horizontal or angular roors and vertical roor centrifuges, usually only measures the absorption value at 260 or 280 mm in length to determine the entire distribution of
nucleic acids
or proteins in the gradient, which is often referred to as on-line monitoring.
.