Condition for sonication of protein solution to get rid of DNA? - How long to sonicate and with what amplitude? (Dec/14/2005 )
I have 80 ml of my periplasmic fluid with overexpressed protein in it. To put in on th Ni column I need to get rid of DNA which makes my colution viscous.
I think the sonication would be the best method. But I have no idea, which should be the interval and intensivity of pulse? I afraid not to denaturate my protein...
I think the sonication would be the best method. But I have no idea, which should be the interval and intensivity of pulse? I afraid not to denaturate my protein...
Hi Alesia,
I usually do 6 intervals of 30 seconds sonicate and 30 seconds rest. Depending on the sensitivity of your protein to go into inclusion bodies, everyone is different. My power is fairly high and duty is about 50%.
Best way for first time...set up microcentrifuge tubes with a little dilute bradford solution in them. Add 1 ul to your 1 ml. Keep sonicating until the solution doesn't get any bluer.
Hope this helps!
Cindy
Thank you very much for your answer.
God Thanks, I have no inclusion bodys, just protein solution, so my aim is just to destroy ADN. For this purpose I think no need to sonicate severely, and Bradfrod test will not help me anymore. But anyway thank you for idea, I realise that no need to sonicate for more than 3 min.
I would suggest to freeze fracture your cells with liquid nitrogen and then precipitate your DNA out of solution. Be sure to use a protease inhibitor cocktail as well.
I do not understand exactly, what I should freeze. I have no cell in solution. I obtain it by using polymixine to perforate the external membrane, than I centrifuge and have my periplasmic fraction. "nude "cells rest in the pellet - they have no interest for me.