Subcellular Fractionation Protocols
Licia Miller Product Manager
Subcellular fractionation is a biological experimental technique that allows researchers to separate and purify different components within cells, such as the nucleus, mitochondria, endoplasmic reticulum, etc., and then further study their structure and function. Centrifugation is one of the most commonly used methods.
Material Required
Subcellular fractionation buffer configuration:
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Before use, add the following to every 10 mL:
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Experiment Steps
(Note that all centrifugation operations should be performed at 4°C. Samples should be kept on ice throughout the process. )
1. Transfer cells from a 10 cm culture plate into 500 μL of separation buffer and incubate on ice for 15 minutes.
1 mL syringe with a 27-gauge needle to pipette the cell suspension up and down 10 times (or until all cells are lysed).
3. Place on ice for 20 minutes.
4. Centrifuge the sample at 720 xg for 5 minutes. The pellet is mainly composed of nuclei, and the supernatant contains cytoplasm, cell membranes, and mitochondria.
5. Transfer the supernatant to a new tube and place on ice. Wait until processing in steps 9-13.
6. Wash the nuclear pellet from step 4 with 500 μL of fractionation buffer : first disperse the pellet with a pipette and pipette up and down 10 times using a 25-gauge needle.
7. Centrifuge again at 720 xg for 10 min. Discard the supernatant and keep the pellet containing the nuclei.Resuspend the pellet in TBS containing 0.1% SDS.
8. Briefly sonicate the suspension to shear genomic DNA and homogenize the lysate ( sonicate on ice for 3 seconds and 2 consecutive settings ).
9. Centrifuge the supernatant recovered in step 5 at 10,000 xg for 5 minutes. The pellet contains the mitochondria. Transfer the supernatant to a new tube and place on ice: this is the cytoplasmic and membrane fraction.
10. Treat the mitochondrial pellet from step 9 as described for the nuclear pellet in step 7 to obtain a mitochondrial lysate in TBS/0.1% SDS.
11. For membrane separation, place the supernatant from step 9 in an ultracentrifuge at 100,000 xg for 1 hour.
12. Wash the pellet by adding 400 μL of separation buffer. Resuspend the pellet with a pipette and filter through a 25-gauge needle. Centrifuge again for 45 minutes. Resuspend the membrane pellet in the same buffer as the nuclei.
13. Depending on the experimental requirements, the supernatant can be further concentrated by centrifugation through a filtration device. This will concentrate the cytosol to approximately 50 - 75 μL.
Subcellular fractionation has a wide range of applications, not only in basic scientific research, but also in disease diagnosis and treatment research. In addition, this technology is also very important in drug development, especially in the field of drug target discovery and new drug development.
When performing subcellular fractionation, there are many factors to consider, including the type of sample, the purity required, the equipment available, and the subsequent analytical methods.
In general, subcellular fractionation is a key technology that plays an important role in modern biomedical research and helps us better understand the complexity of cells and biological processes.
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