Hello,
Currently, I am conducting an experiment using a reporter cell (a mammalian cell expressing target protein in cytosol). The experiment is not going well.
So I would like to check whether the target protein expressed in cytosol is catalytically active.
I'm going to do an assignment in in vitro by extracting target protein(as native form) from the mammalian cell as a troubleshooting. However, I am having a hard time because I don't know how to extract native protein in mammalian cells.
I'm trying to disrupt the cell through sonication, it is difficult to set experimental conditions such as sonication condition(time, amp), concentration of EDTA&protease inhibitor cocktail, etc.
Can you help me with the relevant literature or methods?
Hello Daeseong Kim
Sonication is used to break apart the cell membrane, which releases all proteins into solution. It is the process of applying ultrasonic sound waves to a solution. Over-heating is always a concern when performing sonication because it can damage the protein of interest.
However, sonication is safe for proteins if the sample is kept as cold as possible. It is best to apply the ultrasonic waves in short bursts and to cool the sample on ice between sessions. If the proteins are kept cold enough during sonication, then the process won’t lead to denaturation and loss of higher order protein structure.
I would prefer to use non-denaturing lysis buffer which contains mild non-ionic detergent and a combination of various salts and agents to enhance extraction and stability of proteins. Non-ionic detergent like NP-40 or Triton X-100 are surfactants, that disrupt the fatty acid bilayer. NP-40 can be used to solubilize proteins but not denature them. The non-denaturing buffer should also contain low salt concentration, usually 120 mM NaCl. Some salt is needed to prevent non-specific protein interactions. EDTA which is a common additive has multiple functions including protease inhibition and protection against oxidative damage. It may be added to a final concentration of 5mM. Tris is another additive used to buffer the pH and prevent protein denaturation.
You may prepare a non-denaturing lysis buffer as follows.
NP-40/Triton X-100 lysis buffer:
50 mM Tris HCl, pH 8.5,
120 mM NaCl,
5mM EDTA,
1% detergent (NP40 / Triton X-100).
The concentration of salt and detergent could be altered according to your needs.
Protease inhibitors should be added fresh to the lysis buffer at the time of lysis. You may add protease inhibitor cocktail as 1X working concentration or use a range between 0.1uM to 1mM depending on the type of protease inhibitor.
Hope this helps!
Best.
Thank you very much for your kindness.
I have one more question!
What are the conditions of mammalian cell sonication in general? I am currently using the MDA-MB-231 cell. In the case of E.coli, we did it for 5sec sonication, 15sec rest, and 5min at 40amp, but can we apply the same condition?
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