For the expression of subtle Nus tag fusion protein I have optimized sonication for 5ml culture, 50 percent amplitude, 1pulse off 1pulse ON for 30 second. With repeated cycles with the gap of 1min. It worked well for me but now on the same sonication conditions most of the interest of protein is going into pellet and showing more intense band of interest of protein and less Intense bans was observed in supernatant on Sds gel. Is that because of over sonication?
All things are same expect colony which i used for induction but I used same glycerol stock, only colony is different during Iptg induction.
Kindly suggest why I am getting less Intense band in supernatant?
Hi there,
Your protein of interest looks less soluble (agregated?, membrane associated?) from the start for some reason when you start from different clones... Sonication has nothing to do about it. Oversonication would tend to degrade the proteins. Due to genetic instability of the expression strain, it is often observed the loss or decrease of expression in time. Redo the experiment with freshly transformed cells.
Sonication rarely damages proteins, assuming we are not talking about a very large complexes like eg. ribosomes. It might potentially happen when you overheat your sample.
But it is much more likely that the problems come from the expression process itself. To get more soluble protein you could try lowering temperature (eg.25 instead of 37C) after the induction or lowering the concentration of IPTG used (eg. 0.1 instead of 1 mM).
Sonication is not trivial. I suggest that you test the expression levels by by boiling your cells directly and analyse on SDS-PAGE. Then you will see if it is the sonications step that cause your problem or the actual expression.
In my opinion the best thing that you should try is to keep your sample on ice during the sonication, and even if it takes time try to wait 10 minutes between each sonication cicle. Heat damage is the most destructive thing for proteins during sonication protocols at least in my hands.
Hi Liger...
From the start I used same clone and same glycerol stock as the construct is Nus tag fusion protein so I was getting sufficient expressions in supernatant and less in pellet but suddenly drastic change occured and induced band coming in pellet instead of supernatant.
Something changed in expression: different induction temperature, different culture volume, different iptg amount, etc.
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