Storage of supernatant that contain proteins after cell lysis. - (Oct/20/2008 )

Hi,

My supernatant contain proteins and my protein of interest is an enzyme. After I lyse the cells and collect the supernatant which contain the proteins, how do I store it before I use it to do protein quantification and enzymatic assay?

Can I store at -20 degree celsius? And for how long I can store it?

Thanks in advance!


Regards,
YY

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You can store it in -80°C for long time storage (say about a few months) and in -20°C for shorter storage time (up to some weeks).
This can be used for "normal" proteins, I can't say how long time storage will affect the activity of an enzyme. Perhaps you have to store it with glycerin and maybe Na-Azid?

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I have J774 iNOS enzyme that I isolated 5/6 years ago which is still active. You should always store your samples in lysis buffer with protease inhibitors at - 80 degrees. I have used J774 iNOS enzyme that is 10 years old that has lost most of it's activity BUT is perfect as a positive control in Western blotting.....all stored at -80.
You should also try and aliquot samples like these. Storage is one thing, but MULTIPLE FREEZING AND THAWING will damage your proteins more.

Hope this is useful.

Rhombus

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Thanks for the information!
Actually the supernatant contains the total proteins of the cells, and the enzyme of interest is present. So I'm not isolating pure enzyme. I just need to store it for short period of time, so I think -20 should be fine. Then is it neccesary to add in protease inhibitors into the extraction reagent before adding into the cells to start lysis?

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Actually, even for short periods of time, cell lysates should be stored at -80. The only concern is that freezing the sample will potentially destroy your specific enzymatic activity. I've seen some enzymes survive multiple freeze/thaws without much loss of activity while others are completely dead with a single freeze. Just hope that you are lucky. You need to add protease inhibitors to the cell lysis/extraction buffer BEFORE lysing the cells. I would also recommend some phosphatase inhibitors as well. Many enzymes are activated by phosphorylation and you may loose activity if you don't inhibit dephosphorylation of it.

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Thanks for the input.
If this is the case, so its better to add in protease inhibitor cocktail and phosphatase inhibitor cocktail into the extraction buffer before start lysis to minimise the loosing of enzyme activity. Then I will try to store at -20 and see how's the enzyme activity.

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add glycerol at least 20% end conc to keep it fluid...; very important: fast cooling down

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...and while it won't help this time, if you ever need to do another prep, when you do the cell harvest, try spinning a small volume (say 5%) separately. Put the main cell pellet into the freezer, process the small pellet, and do your assays on it. Then when you know the quantitation and enzymatic assay results, process the remainder.

For this time, if your assays are over within a couple of hours, you should be able to store in ice, then aliquot or purify further as required.

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