Hi everyone,
We are looking for a quick/instant method to perform cell lysis (HEK cells) while monitoring luciferase activity from t=0 to t=5-30 min let say.
So the lysis buffer need to be very strong to instantly rupture the membranes... while keeping protein integrity (and capacity to interact, or not interact). So 30 min on ice / on a wheel in a cold chamber or sonication are out of question.
Hence: osmotic shock. We have tried a (too?) simple PBS 0.1X with some success (luciferase signal up to 1h30 after cell lysis) but I am having cold feet about using that method as I have seen so far very little support in the literature (or even on the world wide web). Should we add some EDTA, Tris, (protease inhibitor)... anything to buffer our solution?
EDIT: i.e. what would be the effect of PBS 0.1X alone on the proteins? what can I do to protect them?
Thank you in advance
Have you considered a nondenaturing detergent, including nonionic and zwitterionic ones, to dissolve the plasma membrane without damaging the luciferase?
Perhaps, you can consider the inclusion of sacharose in your lyses buffer. This actually will depend on whether you expect your proteins on the outer membrane, in the periplasm or the cytoplasm. Saccharose has the ability to confer osmostic protection to the inner membrane; thus, will assist the liberation of proteins in the protoplasm.
I have read a couple of articles which used this strategy in the isolation of protoplasmic and membrane-bound proteins. Some included lysozyme, proteases, EDTA and surfactants. Perhaps, you can improvise that strategy. Check below:
http://www.sciencedirect.com/science/article/pii/S0005273614004398
https://microbialcellfactories.biomedcentral.com/articles/10.1186/s12934-016-0505-8
Do you have any suggestion of nondenaturing detergents? Let say between Tween-20 and Triton X-100 ?
Use Saccharose for cell lysis is new to me. I'm into cytoplasm/nucleus.
But to my understanding, both detergents and saccharose's purpose is to rupture the membrane. My issue is that such chemical approach won't be fast enough compare to an osmotic shock? Or do you imply that they will help protect protein from denaturation and protein-protein interactions?
As I understand it, your goal is to lyse the cytoplasmic membrane quickly so that you can measure the cytoplasmic luciferase activity. This requires a lysis method that is fast, reliable, and does not inhibit luciferase. Since this is a common method, there are kits available that supply the lysis buffer. Here is the manual for one such kit. Triton X-100 is used in the lysis buffer.
https://www.promega.com/-/media/files/resources/protocols/technical-bulletins/0/luciferase-assay-system-protocol.pdf
I mean.... it seems too stupidly simple to be the answer I'm looking for...
But many thanks anyway!
Only question now is how fast does it work....
EDIT: not fast enough
Hi Benoit,
I think you're a bit confused regarding terminology.
'Use Saccharose for cell lysis is new to me. I'm into cytoplasm/nucleus. But to my understanding, both detergents and saccharose's purpose is to rupture the membrane. My issue is that such chemical approach won't be fast enough compare to an osmotic shock?'
First of all, osmotic shock is a cell lysis method, as you also mention in the title of your question. Therefore, any lysis method will result in membrane rupture and release of cytoplasmic components, i.e. what you aim in order to measure the cytoplasmic luciferase activity.
Secondly, saccharose (or sucrose in other words) is an osmotic shock lysis approach, similar to osmotic shock lysis by NaCl.
Third, a mild detergent such as Triton-X or DDM at 1% concentration will result in immediate lysis of mammalian cells such as HEK.
Lastly, you can try various approaches, compare, and see what's best according to your experiment needs. There's no 'magic technique' or 'magic answer'. Both lysis methods (detergent + osmotic shock) result in immediate lysis of HEK cells. Now how many femtoseconds faster is one from another, it's a question that no one can answer I'm afraid. They are both fast (compared to sonication, French press, etc).
You could add in your buffer a protease inhibitor cocktail and/or DNAse in order to degrade your DNA and avoid getting a too viscous sample.
Good luck!
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