I tried 2 extraction methods for mouse liver samples (from ApoE KO mice fed a high fat diet) that had been snap frozen and stored in -80 freezer.
I followed the Abcam sample preparation for tissue lysates and I also tried a protocol that had been posted by a previous reviewer of the product. See details below (copy pasted from previous reviewer):
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.
To homogenise the tissue I used a precellys 24 homogeniser.
Overall the protocols were similar but I preferred the Abcam one as all the reagents required are supplied within the kit. The alternative method requires NP-40 which is not supplied which would be expected.
From the image uploaded you can see that the 2 methods (Method 1 - Abcam Method 2 - user) produced very similar absorbance values (colorimetic method was used). The samples were diluted 5x to ensure they were within the standard curve.
Having compared the 2 methods I will proceed with using the Abcam method.
Overall this is a straightforward easy to use kit.
I followed the Abcam sample preparation for tissue lysates and I also tried a protocol that had been posted by a previous reviewer of the product. See details below (copy pasted from previous reviewer):
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.
To homogenise the tissue I used a precellys 24 homogeniser.
Overall the protocols were similar but I preferred the Abcam one as all the reagents required are supplied within the kit. The alternative method requires NP-40 which is not supplied which would be expected.
From the image uploaded you can see that the 2 methods (Method 1 - Abcam Method 2 - user) produced very similar absorbance values (colorimetic method was used). The samples were diluted 5x to ensure they were within the standard curve.
Having compared the 2 methods I will proceed with using the Abcam method.
Overall this is a straightforward easy to use kit.
The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.
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提交于 Aug 14 2017