Separation of higher components (HDL, VHDL) containing apolipoprotein by electrophoresis is more successful, and centrifugation is suitable for separation of serum lipoproteins of various densities.
Serum lipoprotein is a heterogeneous complex composed of blood lipids and certain specific proteins. Because of the different ratio of lipid to apolipoprotein, the density ranges from 0.96g / ml or lower to 1.21g / ml. Four bands are shown in the paper electrophoresis: milk dense particles, very low density lipoprotein, low density lipoprotein, and high density lipoprotein. Each lipoprotein can also be divided into more subcomponents.
1. Gradient material: The density of serum lipoprotein is low. The commonly used gradient materials are Nacl, NaI, Kbr, KI, etc. The commonly used buffer is ED.
1. Separation of blood cells and serum:
Centrifugal parameters: whole blood, angle rotor, 3,000rpm Ã— 20min
Results: The sediment was blood cells and the upper part was serum.
2. Separation of chyle grains: the content of chyle grains in the plasma of people who have been fasting for more than 12 hours is very small. Those who have eaten should have centrifuged to remove chyle grains because their plasma contains high chyle grains.
Centrifugal parameters: 4 Ã— 10 (g Ã— min), 10 Â° C. Various rotating speeds are available.
Gradient configuration: add 3/4 volume in the lower part of the centrifuge tube with plasma, and add 0.5MnaCl 0.3MEDTA, ph7.4 in the upper 1/4 volume
Result: The chyle grains floated up and sucked out.
3. Serum protein separation: remove globulin, albumin and other proteins.
Centrifugal parameters: (2.5-5) Ã— 10 (g Ã— hr), all kinds of rotors are available. For example, corner rotating head 70,000rpm Ã— 6hrs, 10 Â° C
Configuration: 1/3 of the tube volume is serum, 2/3 is 1.31g / ml, NaCl NaBr, the final density after stirring is 1.21g / ml
Results: The upper 1/6 volume of the tube was serum lipoprotein and the lower 5/6 was other proteins.
1. Sequential flotation method:
a) Overspeed method: the earliest used method, 4000rpm Ã— 24hrs Ã— 3 times, 10 Â° CNaCl NaBr gradient flotation in order of 1.006, 1.063, 1.21. The different density of the upper part of the tube is taken out
lipoprotein. Simple operation, high resolution, high yield, time-consuming and high cost.
b) Ultra-high speed method: 100,000rpm Ã— 2.5hrs Ã— 2 times, 10 Â° C, 100,000rpm Ã— 4hrs Ã— 1 time, NaCl KBr EDTA gradient flotation VLDL, LDL, HDL according to no density
2. Single centrifugation method:
a) Single horizontal separation
With a maximum rotation speed of 25,000 ~ 28,000rpm, a 6 Ã— 40ml flattening rotor with 1.006g / ml NaCl solution and 1.35g / ml (NaCl KBr EDTA) solution is configured into 8 layers from 1.06 to 1.21. Do not
Continuous step, the lowest part is prepared with serum and KBr to 1.25g / ml sample solution, 25,000rpm Ã— 4hrs, 4oC to get different layers of VLDL, LDL, HDL at one time.
With a maximum rotation speed of 40,000rpm, 6 Ã— 13ml swing-out rotor, 40,000rpm Ã— 24hrs, 20 Â° C, discontinuous KBr gradient 1.006 ~ 1.21g / ml, elongated tube, clear layering, high recovery rate, centrifugal long time
b) Single vertical tube separation:
NaCl / KBr or NaCl / NaBr discontinuous gradient single tube capacity from 5ml to 40ml, rotation speed from 50,000rpm to 80,000rpm, centrifugation (0.5-3) hrs, lower layer with KBr or (NaBr) to adjust serum to 1 .3g / ml upper layer with 1.006g / ml, NaCl solution, 10 Â° C-20 Â° C, increase, decelerate once to separate. The lower layer of NaCl / Sucrose discontinuous gradient is 48% W / WSucrose, the middle layer serum is in 4MnaCl, and the upper layer is 0.67MNaCl 0.05% EDTA.
c) Large-capacity separation of zone rotor:
Centrifugal parameters: zone rotor, maximum speed 30,000 ~ 48,000rpm, capacity from 650ml ~ 1900ml, serum can be separated 50-150ml each time, low speed sample loading and unloading, separation speed: large rotor
(25,000-28,000rpm) Ã— 24hrs, small rotor (30,000-38,000rpm) Ã— 1hrs, 10 Â° C-20 Â° C
Gradient solution: NaCl NaBr or KBr EDTA discontinuous or linear gradient, the sample is in the maximum density layer, near the core of the rotor with pure water as the isolation layer, and the outermost part of the separation chamber uses high density NaCl / KBr or NaCl / NaBr as the buffer layer.
Collection: After being pumped out, it will be detected by a UV detector with a flowing place, and then automatically collected by a partial collector.
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