In order to extract protein from my cell lines I always used to destroy the cell lysate using syringe. After centrifugation I used to got a good pellet (cell debris and nuclear protein) in the bottom and cytoplasmatic and membrane proteins from the supernatant (in the top). After, I proceeded to extract nuclear proteins adding other detergent.
I changed a little my protocol using sonification instead syringe to destroy the cells. But after centrifugation (13 mins, 12 000 rpm) I noticed a VERY little pellet and in many case no pellet. I would know if does sonification process has destroyed to much (if yes, I would not be able to get separately nuclear OR cyto/memb proteins)? and 2) Should I draw up the supernatant (to get cyto and memb proteins) as I used to do before?
Thank tou!
I have performed sonification for extraction of nuclear and cytoplasmic proteins and have been successful.
I don't know what protocol are you following, but the one that i use requires a very slow speed for extraction of cytoplasmic protein which i do by the conventional way; i.e ice incubation following gentle vortexing for 20 mins and extract the supernatant using a speed of 3000-5000 rpm for 3-5 mins.
This step should be gently performed!
Then i wash it with washing buffer almost 4-5 times to wash off the cytoplasmic content and everytime use the centrifuge at a speed of 4000-5000 foe 3-5 mins and finally for the extraction of nuclear protein, i use sonification ( generally the protein conc of nuclear fraction is very weak by conventional extraction technique but it increases with sonification. However, sonification should be done for few secs with a rest period to prevent heating of the tip and destroying of protein.
Hope it helps!
in sonication process generate huge heat that mey cause degradation of your protein but sonication not
Dear Dr. Madhu, thank you so much for your answer. I could checked, today, that using sonification process immediately after I scrached cells from the flask i would just able to get the proteins quantity of whole cells (nuclear + cytoplasmatic proteins). Next time, as soon as I will need just the nuclear or cyto proteins fraction I would prefer use your protocol which consists in two different steps.
Thank you to Dr. arvind for the advice too.
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