I have been doing a subcellular fraction of Nucleus, Mitochondria, and ER in the HEK293 cell. But in results, I usually see all the three fractions have an equal amount of protein. Please help me to sort out the protocol.
hello Simranjit Singh what do you mean by "equal amount of protein" ? to test the specificity of your fractionation protocol you should do Western blot of the three fractions and probe for fraction specific proteins. example VDAC for mitochondria, Calreticulin for ER, and lamin A+C for nuclear fraction. these should be concentrated in their respective fractions but not in the other fractions. hope this helps
Hello Simranjit Singh ,
There are different protocols at your disposal for this experiment. I can link a couple of them here which worked for me:
1) Article Crude subcellular fractionation of cultured mammalian cell lines
2)Article A simple protocol for the subcellular fractionation of skele...
As Rabeah suggested, you should also double check the efficiency of your procedure by blotting for markers for the different subcellular compartments.
Good luck with your fractionation.
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