TISSUE PREPARATION
For total cholesterol
1. Homogenize 10 mg tissue in 200 μl of chloroform:Isopropanol:NP-40 (7:11:0.1) using a micro-homogenizer.
2. Spin the extract 10 minutes at 15,000 x g at RT in a centrifuge.
3. Transfer all of the liquid (organic phase) avoiding the pellet, to a new tube. Air dry the sup at 50°C to remove chloroform for 7min (in a hood).
4. Dissolve dried sup with Cholesterol Assay Buffer (1:1) by vortexing until homogeneous (it is OK if the solution becomes cloudy).
Note: The extraction procedure can be scaled up if larger amounts of sample are desired.
For the separation of HDL and LDL/VLDL
1. Use 0.5% NP-40 in water to homogenize the tissue and then combine with the 2X precipitations buffer.
2. Mix 100 µL of sample with 100 µL of 2X Precipitation Buffer (1:1) in microcentrifuge tubes.
3. Incubate 10 minutes at room temperature.
4. Centrifuge at 2,000 x g (5,000 rpm on a bench-top microcentrifuge) for 10 minutes.
5. Transfer the supernatant into new labeled tubes. This is the HDL fraction.
6. Precipitates contain the LDL/VLDL fraction. To measure the LDL/VLDL fraction, centrifuge the precipitate again at 2,000 x g (5,000 rpm on a bench-top microcentrifuge at RT) for 10 minutes and remove trace amount of HDL supernatant carefully. I tissue homogenate you cannot measure the LDL fraction, only HDL!!!! (In tissue culture it is possibale).
7. Resuspend the precipitate in 200 µL PBS. This is the LDL/VLDL fraction.
If the supernatant is cloudy, the sample should be re-centrifuged. If the sample remains cloudy, dilute the sample 1:1 with PBS and repeat the separation procedure from step 2.
For total cholesterol
1. Homogenize 10 mg tissue in 200 μl of chloroform:Isopropanol:NP-40 (7:11:0.1) using a micro-homogenizer.
2. Spin the extract 10 minutes at 15,000 x g at RT in a centrifuge.
3. Transfer all of the liquid (organic phase) avoiding the pellet, to a new tube. Air dry the sup at 50°C to remove chloroform for 7min (in a hood).
4. Dissolve dried sup with Cholesterol Assay Buffer (1:1) by vortexing until homogeneous (it is OK if the solution becomes cloudy).
Note: The extraction procedure can be scaled up if larger amounts of sample are desired.
For the separation of HDL and LDL/VLDL
1. Use 0.5% NP-40 in water to homogenize the tissue and then combine with the 2X precipitations buffer.
2. Mix 100 µL of sample with 100 µL of 2X Precipitation Buffer (1:1) in microcentrifuge tubes.
3. Incubate 10 minutes at room temperature.
4. Centrifuge at 2,000 x g (5,000 rpm on a bench-top microcentrifuge) for 10 minutes.
5. Transfer the supernatant into new labeled tubes. This is the HDL fraction.
6. Precipitates contain the LDL/VLDL fraction. To measure the LDL/VLDL fraction, centrifuge the precipitate again at 2,000 x g (5,000 rpm on a bench-top microcentrifuge at RT) for 10 minutes and remove trace amount of HDL supernatant carefully. I tissue homogenate you cannot measure the LDL fraction, only HDL!!!! (In tissue culture it is possibale).
7. Resuspend the precipitate in 200 µL PBS. This is the LDL/VLDL fraction.
If the supernatant is cloudy, the sample should be re-centrifuged. If the sample remains cloudy, dilute the sample 1:1 with PBS and repeat the separation procedure from step 2.
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提交于 Mar 29 2016