Centrifugal separation method and characteristics of high speed centrifuge
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A. Differential centrifugation: The centrifugal force is increased successively, and some components in the sample solution can be settled each time.
Differential centrifugation is a common method used by Zui. In this method, the centrifuge tube is full of a uniform sample solution at the beginning of the fashion. After centrifugation at a certain speed for a certain period of time, two fractions are obtained: the precipitate and the supernatant.
Most of the unwanted large particles are usually removed by centrifugation at * times. Most of the components required at this time remain in the supernatant. The collected supernatant is then centrifuged at a higher speed to deposit the desired particles. The time of centrifugation should be chosen so that most of the smaller particles that are not needed remain in the supernatant. The resulting precipitate and supernatant can be subjected to further centrifugation until the desired separation purity is achieved.
Differential centrifugation is characterized by simple operation, but the purity of the separation is not high.
B. Density Gradient Centrifugation: It is possible to separate several or all components of a sample at the same time with good resolution.
(1) Rate zone method (ratezonal):
Depending on the size and settling velocity (S) of the different particles in the sample. The general steps are as follows:
A density gradient solution was placed in the centrifuge tube, and the density of the solution gradually increased from the top to the bottom of the centrifuge tube (positive gradient).
The desired separated sample was carefully added to the top of the density gradient solution. The sample forms a negative gradient on the surface of the gradient solution.
Since different sized particles move at different speeds in the gradient under centrifugal force, several separate sample zones are formed after centrifugation.
Note: The density of the sample particles must be greater than the density at any point in the gradient. The centrifugation process must be stopped before the zone reaches the bottom of the tube.
(2) Equal density centrifugation (isopycnic):
Separate according to the different densities of the particles. During centrifugation, the particles move to a zone of the same density as itself.
The density is chosen such that the range of gradients includes the density of all particles to be separated. The sample can be above the density gradient liquid particles or evenly distributed in the density gradient. After centrifugation, the sample particles reach their equilibrium point.
Note: The separation of particles after equilibrium is completely determined by its density, regardless of time. At this time, changing the centrifugal speed can only change the relative position of the zone.
Density Gradient Analysis (1) Gradient Media Properties and Options:
A. What should be possessed:
The principle of selection of gradient materials is to meet the basic requirements of the separation method. An ideal density material standard should be:
The density of the solution formed should include the range of densities required.
It has certain properties, such as refractive index, from which its concentration can be determined.
The resulting solution has a low viscosity.
The separated sample is not damaged.
It is easy to remove after centrifugation.
Does not hinder the analysis of the separation integral.
B. Common media types:
Table 1, commonly used gradient materials at 20 ° C density B. Gradient media application range:
Table 2, Application of Equal Density Gradient Media Table 3. Approximate Density of Various Macromolecules in Sucrose Gradient Solution (2), Preparation of Gradient Solution:
Calculate, dilute (3) gradient shape gradient shape points: linear, constant velocity, stepped, flat, steep exponential gradient.
The gradient shape is very important for the success of the separation:
Zui is commonly used in linear gradients, which are suitable for separating proteins, enzymes, hormones, ribosomal subunits and some plant viruses; isokinetic is suitable for separating lipoproteins and some samples that need to be separated by floating; discontinuous or stepped gradients are suitable for Isolation of whole cells, subcellular components, and purification of some mammalian or insect viruses. The isokinetic gradient and long liquid column enhance separation and are suitable for the separation of ribosomal subunits, polysomes and plant viruses.
B. Gradient column preparation:
Gradient liquid column can be prepared by hand or gradiometer:
In order to reduce the centrifugation time, or to separate the sample, it is less feasible to use the half-injection method: the lower half tube is placed with a gradient medium, and the sample is added in the middle, and Buffer or liquid paraffin oil is placed on top.
(4) Sample loading method and sample loading:
Add the sample to the gradient column. The tip and the tube are at an angle of 45-60°. Slowly flow the sample along the tube wall to the liquid surface. For vulnerable samples such as DNA, the aperture should be larger. Pipettes replace the needle to avoid shearing of the sample by shear forces. The sample concentration is 1/10 (W/W) of the small density of the gradient column zui.
(5) Selection and effect of the rotor:
(6) Recovery of separation zone and detection There are basically four methods for recovery of zone samples formed after centrifugation:
a. Puncture the bottom of the centrifuge tube, let the gradient solution drip out, and place a cap with a suitable valve on the top of the centrifuge tube to control the dripping speed.
b. Siphon method:
Gently insert a capillary into the bottom of the tube to prevent gradient jitter. Gradly draw with a micro pump and collect it in a certain amount of droplets or volume.
c. Pressurization method:
A high-density liquid is pumped through a needle into the bottom of the gradient tube to partially collect the exchanged solution.
d. Cutting method:
The required zone is cut with a dedicated cutter.
Zone detection:
The so-called zone detection is actually the monitoring of the separated substances in the horizontal or angular rotor and the vertical rotor centrifuge tube. Usually, the absorption value at 260 or 280 mm is usually measured to determine the whole nucleic acid or protein in the gradient. Distribution, this operation is often referred to as online monitoring.