Preparation of lysis buffer for enzyme extraction - (Feb/25/2008 )

Hi, is lysis buffer made up of 0.1 M sodium phosphate buffer (pH 6.8) containing 1% triton X-100 and 1% protease inhibitor cocktail suitable to be used for enzyme extraction? Should I prepare the lysis buffer immediately when want to use or can stored in the fridge until use?

Is freeze-thawing cycle necessary to extract the enzyme effeciently?

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we prepare the buffer for lysis which is stored in 4C. But we add protease inhibitors fresh just before using it.

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How much concentration of protease inhibitors you used?

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we have 2 different PI, one of them from a tablet cocktail we use as 100x and other as 1000x.

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If I buy the powder form protease inhibitor cocktail (1 botol makes 100 ml of cocktail), this 100 ml will be my stock solution and I just add into lysis buffer immediately when I want to lyse the cells? Is 1 % of protease inhibitor cocktail in 100 ul of lysis buffer enough for cell lysis in 96-well plates?

Does the phosphate buffer (pH6.8) need to be sterilised before we add in the triton X-100 and protease inhibitor coktail?

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Lets say if you have 20 wells (96 wells plate), take out 3 ml of lysis buffer, add 30ul of this PI cocktail you have and mix it. This should be fine.

We don't sterilize buffer for extracts. Buffer can be made with triton and stored in 4C. Add PI immediately before use.

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