Several separation methods for preparative ultra-high-speed centrifuges

Several separation methods of the preparative ultra-high-speed centrifuge: A.
Differential centrifugation: increasing the centrifugal force one by one, each time some components in the sample solution can be settled
.

Differential centrifugation is a commonly used method
.

In this method, the centrifuge tubes are filled with a uniform sample solution
at the beginning.

After centrifugation at speed time, two parts can be obtained: the precipitate and the supernatant
.

Most of the unwanted large particles are usually removed
during the subcentricion.

At this time, most of the required components remain in the supernatant
.

The collected supernatant is then centrifuged at a higher rate to deposit the desired particles
.

The centrifugation time should be properly chosen so that most of the unwanted smaller particles remain in
the supernatant.

Further centrifugation of the resulting pellet and supernatant can be performed until the desired separation purity is achieved
.

Differential centrifugation is characterized by simple operation but low
separation purity.

B.
Density gradient centrifugation method: several or all components in the sample can be separated at the same time, with good resolution
.

1) Rate zonal method: according to the different sizes and sedimentation rates (S)
of different particles in the sample.

The approximate steps are as follows: Load the centrifuge tube with a density gradient solution, and the density of the solution gradually increases from the top to the bottom of the tube (positive gradient
).

Carefully add the samples 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 differently in the gradient under the action of centrifugal force, several separate sample bands
are formed after centrifugation.

Note: The density of the sample particles is greater than the density
at either point in the gradient injection.

The centrifugation process stops
before the zone reaches the bottom of the tube.

2) Isopycnic: Separation
according to the different densities of particles.

During centrifugation, the particle moves to the same density as it itself to form a zone
.

The choice of density sample size makes the range of the gradient include the density
of all particles to be separated.

Samples can be on top of density gradient particles or evenly distributed in density
gradients.

After centrifugation, the sample particles reach their equilibrium point
.

Note: The separation of particles after equilibrium is determined by their density and is independent of time, at this time, changing the centrifugal speed can only change the relative position
of the zone.

2.
Density gradient analysis method 1) Gradient medium properties and selection: A, should have the properties: the selection principle of gradient substances is to meet the basic requirements of the separation method, an ideal density material standard It should be: · The density of the resulting solution should include the desired range of
densities.

· It has certain properties, such as refractive index, on which its concentration
can be determined.

· The resulting solution has a low
viscosity.

· Does not damage the sample being separated
.

· It is easy to remove
after centrifugation and separation.

· Does not hinder the analysis of the separation integral.
B, commonly used media types: Table 1, common gradient materials in 20 °C density B.
Gradient medium application range: Table 2, the application of isometric gradient media +++ is very good ++ good + can -- not applicable Table 3, the approximate density of various macromolecules in sucrose gradient liquid (2), preparation of gradient solutions: calculation Dilution (part 3 of this article) (3), gradient shape Gradient shape is divided into: linear, isamic, stepwise, flat, steep exponential
。
Gradient shape is important for successful isolation: Linear gradients are commonly used and are suitable for the separation of proteins, enzymes, hormones, ribosome subunits and some plant viruses; The isachronous type is suitable for isolating lipoproteins and some samples that need to be floated up; Discontinuous or stepped gradients are suitable for isolating whole cell and subcellular components and for purifying some mammalian or insect viruses
.

The ismuthrometric gradient and long liquid column can improve the separation capacity and are suitable for the separation of ribosome subunits, polysomes and plant viruses
.

B.
Gradient column preparation: Gradient liquid column can be prepared by hand or gradient meter Half injection method: To reduce the centrifugal time, or to separate the sample less, the half injection method can be used: the lower half of the tube is laid with gradient medium, the sample is added in the middle, and the top is covered with Buffer or liquid stone wax oil
.

(4), the dosing method and the amount of dosing: the sample is added to the gradient liquid column, the needle tip and the centrifuge tube at a 45-60 ° angle, slowly flow the sample along the tube wall to the liquid surface, for DNA and other fragile samples that are easy to break, the needle should be replaced by a pipette with a larger pore size to avoid the cutting effect
of shear force on the sample.

The sample concentration is 1/10 (W/W)
of the small density of the gradient column.

(5), the selection and effect of the rotor: (6), the recovery and detection of the separation zone There are basically four kinds of recovery methods for the strip sample formed after centrifugation: a.
Puncture method Puncture the bottom of the centrifuge tube, so that the gradient solution drips out, and a cap with a suitable valve is placed on the top of the centrifuge tube, which can control the dripping rate
.

b.
Siphon method: Gently insert a capillary tube into the bottom of the tube, try to prevent gradient jitter, and gradually drip with a micro pump to collect
the number of drops or volume.

c.
Pressurization method: The high-density liquid is pumped into the bottom of the gradient centrifuge tube through a syringe tube, and the swapped solution
is partially collected.

d.
Cutting method: Use a special cutting knife to cut the required area.
Zone detection: The so-called zone detection is actually the monitoring of separated substances in horizontal rotors or angle rotors and vertical rotor centrifuge tubes, usually only measuring the absorption value at 260 or 280 mm to determine the entire distribution of nucleic acids or proteins in the gradient, this operation is often called on line monitoring
.

How to properly maintain your centrifuge Centrifuge Development centrifuges are instruments that separate samples, widely used in biomedicine, petrochemical, agriculture, food hygiene and other fields
.

Since the advent of centrifuges, it has undergone changes of low speed, adjustment and overspeed, and its progress is mainly reflected in centrifugal equipment and centrifugal technology, which complement each other
.

From the perspective of speed, tabletop centrifuges basically belong to the category of low-speed and high-speed centrifuges, and the development of general-purpose desktop centrifuges has blurred the boundaries of low-speed, high-speed, micro- and large-capacity centrifuges, and many rotors provide researchers with a fairly wide range of applications and become scientific research laboratory models
.

Centrifuge principle Using centrifugal force, according to the different principles of sedimentation coefficient, mass, density, etc.
of each component in the mixture, the liquid and solid particles in the sample mixture or the liquid and liquid are separated
.

Centrifuge classification Centrifuges are very common separation instruments in laboratories can be divided into low-speed centrifuges, high-speed centrifuges, ultracentrifuges, etc.
according to the speed; Normal centrifuge: speed less than 3500r/min High-speed centrifuge: speed between 3500 r/min ~ 50000 r/min Ultra-high-speed centrifuge: speed greater than 50000 r/min
.

According to the temperature, it can be divided into refrigerated centrifuge and normal temperature centrifuge; According to the capacity, it can be divided into microcentrifuge, large capacity centrifuge, super capacity centrifuge; According to the appearance, it can be divided into tabletop centrifuge and floor-standing centrifuge
.