Separation technique of serum lipoprotein

Serum lipoproteins are a heterogeneous complex of lipids in the blood that are composed of certain proteins. Due to the difference in the ratio of lipid to apolipoprotein, the density ranges from 0.96 g/ml (or lower) to 1.21 g/ml. Four bands are shown in paper electrophoresis: milk dense particles, very low density lipoprotein (VLDL, also known as pre-beta lipoprotein), low density lipoprotein (LDL, also known as beta lipoprotein), high density lipoprotein (HDL, Also known as alpha lipoprotein). Each lipoprotein can also be divided into more sub-components.

Separation of higher apolipoprotein-containing components (HDL, VHDL) by electrophoresis was successful, while centrifugation was suitable for separation of serum lipoproteins of various densities.

1 gradient material:

The serum lipoprotein density is low. The commonly used gradient materials are Nacl, NaI, Kbr, KI, etc. The commonly used buffer is EDTA.

Table 1 Configuration and Density of Common Gradient Materials (20 ° C)

153gNaCl+354gKBr+10ml

0. 2mol / L EDTA + water to a total of 1000ml

NaCl, NaBr

Solution A: 200g NaCl + 800ml water

Solution B: 400g NaBr + 600ml water

10ml A solution + 31.7ml B solution

10ml A solution + 15.6ml B solution

4ml C solution + 6ml 1.35 solution

2 pre-separation:

2.1 Separation of blood cells from serum:

Centrifugal parameters: whole blood, angular head, 3000 rpm × 20 min

Results: The precipitate was blood cells and the upper part was serum.

2.2 Separation of chylomicrons: The chylomicron content in human plasma is very low after fasting for more than 12 hours. Those who have eaten have high chylomicron in their plasma, and should first centrifuge to remove chylomicron.

Centrifugal parameters: 4 × 106 (g × min), 10 ° C, all kinds of rotor speed can be.

Gradient solution configuration: 3/4 volume of the lower part of the centrifuge tube plus plasma, upper 1/4 volume plus 0.15 mol/L NaCl + 0.3 mol/L EDTA, pH 7.4

Result: The chylomicrons floated up and sucked out.

2.3 Separation of serum proteins: removal of globulin, albumin and other proteins.

Centrifugal parameters: (2.5-3) × 106 (g × hr), all kinds of rotors can be. Such as angular turning head 70,000 rpm × 6hrs, 10 ° C

Configuration: tube capacity 1/3 is serum, 2/3 is 1.31g/ml, NaCl+NaBr, and the final density after stirring is 1.21g/ml

Results: 1/6 of the upper volume of the tube was serum lipoprotein, and the lower 5/6 was other proteins.

3 methods:

3.1 sequential flotation method:

3.1.1 Overspeed method: the earliest method used, 40,000 rpm × 24 hrs × 3 times, 10 ° C

The NaCl+NaBr gradient was floated in the order of 1.006, 1.063, and 1.21, and lipoproteins of different densities were taken out at the top of the tube each time. The operation is simple, the resolution is high, the yield is high, time-consuming and costly.

3.1.2 super high speed method: 100000 rpm × 2.5 hrs × 2 times, 10 ° C, 100000 rpm × 4 hrs × 1 time, NaCl + KBr + EDTA gradient flotation VLDL, LDL, HDL

3.2 single centrifugation method:

3.2.1 Single split separation

With a maximum speed of 25000~28000 rpm, 6×40ml 甩 flat head is configured with 1.006g/ml NaCl solution and 1.35g/ml (NaCl+KBr+EDTA) solution to form 8 layers of discontinuous steps from 1.006 to 1.21. The lowermost serum was combined with KBr to form a 1.25 g/ml sample solution, 25000 rpm×4 hrs, and VLDL, LDL, and HDL different layer zones were obtained once at 4 °C.

With a maximum speed of 40,000 rpm, 6 × 13ml 甩 flat head, 40,000 rpm × 24hrs, 20 ° C, discontinuous KBr gradient 1.006 ~ 1.21g / ml, slender tube, clear layer, high recovery, long centrifugation time

3.2.2 Single vertical tube separation:

The NaCl/KBr or NaCl/NaBr discontinuous gradient single tube capacity is from 5ml to 40ml, the rotation speed is from 50,000 rpm to 80000 rpm, centrifugation (0.5-3) hrs, and the lower layer is adjusted to the upper layer of 1.3g/ml with KBr or (NaBr). With 1.006 g/ml, NaCl solution, 10 ° C - 20 ° C, increase, decelerate once and separate.

The lower layer of NaCl/sucrose was 48% W/W sucrose, the middle serum was 4mol/L NaCl, and the upper layer was 0.67M NaCl+0.05% EDTA. The centrifugation parameters were the same as above, and the time was longer.

3.2.3 zone with rotor large capacity separation:

Centrifugal parameters: zone rotor, the highest speed is 30,000~48000rpm, the capacity is from 650ml~1900ml, the serum can be separated 50-150ml each time, low speed loading and unloading, separation speed: large turning head (25000-28000rpm) × 2hrs, small Rotor (30,000-38,000 rpm) × 1hrs, 10 ° C - 20 ° 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, the outermost part of the separation chamber is high density NaCl / KBr or NaCl / NaBr As a buffer layer.

Collection: After pumping out, it is detected by the UV detector with the flow of the belt, and the automatic part collector collects.

4 Discussion:

1. The VLDL, LDL, and MDL separated by the above method can further reduce the discontinuous gradient density difference by using a long centrifuge tube to separate the subcomponents.

2. The quality of the experiment depends largely on the density calibration. After the configuration, it can be checked by a density agent or a refractometer.

3, ultra-analytical ultracentrifugation method can accurately separate trace serum lipoprotein, and can determine the floating coefficient Sf of various components (including its sub-components).