For best results Urea Lysis Buffer should be prepared fresh prior to each experiment when working with PTMScan® protocols.
To make Urea Lysis buffer:
1. Add 45 ml of reverse osmosis deionized (RODI) or equivalent water and 10 ml of 200 mM HEPES buffer (#44686) and mix well until the urea goes completely into solution.
2. Add 1 ml of 100X stock of fresh Phosphatase Inhibitor Cocktail (#5870) just before use. Measure the volume and bring up to the final volume of 100 ml with RODI water.
Note: Dissolving urea is an endothermic reaction that decreases the temperature of the solution. This reaction will cause the urea to precipitate. Allow the solution to warm to room temperature to ensure all of the urea dissolves. The cell lysis step should take place at room temperature to prevent the urea from precipitating out of solution.
See PTMScan® protocols for additional details.
1X Urea lysis buffer is 9M Urea at 100 ml final volume.
Store at room temperature. The final buffer solution can be stored for up to 2 months at -80°C.
Urea is an organic compound that is highly soluble in water with strong protein denaturing properties. These properties make it ideal for use in preparation of Urea Lysis buffer for preparing cell and tissue extracts prior to PTMScan®-based applications. Urea, Ultrapure, PTMScan® Qualified from Cell Signaling Technology is offered in a convenient 54.05g size that allows for the preparation of 100ml of Urea Lysis Buffer with a final formulation of 20mM HEPES, 9M urea, and 1X Phosphatase Inhibitor Cocktail when following the Directions for Use.
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