Monday, October 27, 2003
Description CPP32 Activity Assay Using CPP32 Fluorogenic Substrate Procedure . Determine the protein concentration of the sample to be analyzed (see protocol on Quantification of Protein Samples). For each reaction (prepared in triplicate for each sample): 2. To small glass vials, add between 10 to 30 g of protein per reaction well plus 10% (see Hint #1). 3. Add Lysis Buffer to a final volume of 445.5 l. 4. Add 198 l of Substrate Buffer. 5. Add 16.5 l of 1600 M Substrate Stock and vortex. 6. The final reaction volume should be 660 l. 7. Incubate at 37C for 1 hour. 8. Pipette 200 l from each reaction volume and distribute into a 96-well plate (see Hint #2). 9. Determine fluorescence at an excitation wavelength of 365 nm and an emission wave length of 450 nm. Recipes Lysis Bufer 50 mM Tris 150 mM NaCl 0.5 mM EDTA 0.5% NP-40 pH 7.5 Substrate Buffer 1 part 50 mM DTT 5 parts HEPES/NaCl Stock Buffer Substrate Stock Store at -20C 1.6 mM Apopain/CPP-32 Substrate in DMSO (CAUTION! see Hint #1) 50 mM DTT HEPES/NaCl Stock Buffer 0.2 M NaCl 40 mM HEPES pH 7.5 Supplies Tips 1. For example, for 10 g of protein per reaction well add 11 g to the reaction well). 2. Each reaction should be analyzed in triplicate. Thus each sample is reacted in triplicate and each reaction is analyzed in triplicate. (责任编辑:泉水) |