With the use of different-capacity angle rotors and easily prepared gradient fluids, satisfactory separation results can be obtained on various types of ultracentrifuges. Here are a few examples of separation, in fact, it can also be applied to fixed angle rotors of various models and capacities.
(1) Gradient fluid
Liquid A: 11.40g Nacl, 0.1g EDTA-Na2, placed in a 1000ml measuring cylinder, add 500ml of distilled water and 1
ml 1N NaOH, mix until all are dissolved. Increase the weight of distilled water to 1000ml and finally add 3ml of distilled water (Nacl: 0.195mol). The final density is 1.006g / cm3, PH10.37.
Solution B: Add 24.98g NaBr to 100ml Solution A. The final density is 1.182g / cm3. (Nacl: 0.195 mol, NaBr: 7.65 mol), PH 9.72.
Solution C: Add 78.32g NaBr to 100ml Solution A. The final density is 1.478g / cm3. (Nacl: 0.195 mol, NaBr: 7.65 mol), PH 8.86.
(2) Centrifuge and rotor
• Centrifuge: Hitachi CP-MX series ultra-speed centrifuge or other similar units.
Hitachi CS-150GXL micro ultracentrifuge.
• Rotating head, centrifuge tube; S 140 AT: 140,000rpm, 1,050,000 × g, 10 × 2ml, K = 5, 1PC thick-walled tube.
S 80 AT3: 80,000 rpm, 415,000 × g, 8 × 8 ml, K = 23, 6PC thick-walled tube.
S 70 AT: 70,000 rpm, 505,000 × g, 8 × 40ml, K = 44, 40PA tube.
(3) Plasma-hemocyte separation:
Whole blood, turn the corner or turn the rotor at 3000rpm × 20min, precipitate as blood cells, and the supernatant plasma to be used.
(4) Separation of chyle grains:
• Centrifugal parameters: all kinds of rotors are available, 4 × 106 (g × min), 10 ℃
• Gradient fluid: the lower 3/4 volume of the centrifuge tube is plasma, and the upper 1/4 volume is 0.15MNacl + 0.3MEDTA. PH7.4.
• Result of centrifugation: chyle grains float upward, the centrifuge tube is inclined, and the straw is slowly sucked out against the wall of the pipette (approximately 2 to 6% of the plasma volume. Blood content of chyloplasm in blood donors who are banned for more than 12 hours is very low, and the content of blood donors after eating is higher.
(5) Separation of serum lipoprotein:
(â… ) VLDL separation (Ï <1.006g / cm3)
Turn head
Liquid distribution in the centrifuge tube
Centrifugal parameters
result
S140AT
(CS-150GXL unit)
Lower 600μl plasma
Upper 300μl A solution
140,000rpm × 50min
16 ℃
Speed ​​up: 5
Deceleration: 7
It can be seen that about 1/3 of the upper part of the obvious layer is sucked out
S80AT3
(CS-150GXL unit)
Lower 3.4ml plasma
Upper 1.7mlA liquid
80,000rpm × 4.5hrs
16 ℃
Speed ​​up: 5
Deceleration: 7
Same as above
P70AT
(CP-MX unit)
Lower 24ml plasma
Upper part 11.7ml A liquid
70,000rpm × 8hrs
16 ℃
Speed ​​up: 5
Deceleration: 7
Same as above
(Ii) LDL (including IDL) separation (1.006g / cm3 <Ï <1.063g / cm3)
Turn head
Liquid distribution in the centrifuge tube
Centrifugal parameters
result
S140AT
(CS-150GXL unit)
After the first centrifugation, the upper VLDL is taken out, the lower 600μl is transferred to another centrifuge tube, 300μl B solution is added, and mixed well
140,000rpm × 80min
16 ℃
Acceleration: 9 Deceleration: 7
Upper 1/3 volume LDL + IDL suction
S80AT3
(CS-150GXL)
After the first centrifugation, the upper VLDL is sucked out, and the lower 3.4ml is transferred to another centrifuge tube, 1.7ml of B solution is added and mixed thoroughly
80,000rpm × 5.5hrs
16 ℃
Acceleration: 9 Deceleration: 7
Same as above
P70AT
(CP-MX series speeding machine or similar models)
After the first centrifugation, 11.7ml of the upper part is sucked out, about 24ml of the lower part is transferred into another centrifuge tube, and 11.7ml of B liquid is added, and mixed thoroughly
70,000rpm × 10hrs
16 ℃
Acceleration: 9 Deceleration: 6
Same as above
(â…¢) HDL separation (1.063g / cm3 <Ï <1.21g / cm3)
Turn head
Liquid distribution in the centrifuge tube
Centrifugal parameters
result
S140AT
(CS-150GXL unit)
After the second centrifugation, aspirate 300 μl of the upper part, 600 μl of the lower part into another centrifuge tube, add 300 μl of C solution, and mix well
140,000rpm × 140min
16 ℃
Speed ​​up: 9
Deceleration: 7
Visible stratification,
The upper part is HDL, the lower part is serum albumin and globulin
S80AT3
(CS-150GXL unit)
After the second centrifugation, aspirate about 1.7ml of the upper part and 3.4ml of the lower part into another centrifuge tube, add 1.7ml of C solution and mix well
80,000rpm × 8 hours
16 ℃
Speed ​​up: 9
Deceleration: 7
Same as above
P70AT
(CP-MX series machine or other types of units)
After the second centrifugation, aspirate the upper 1/3 and lower 2/3 into another centrifuge tube, add about 12ml of C solution, and mix thoroughly
70,000rpm × 15 hours
16 ℃
Speed ​​up: 9
Deceleration: 6
Same as above
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