EricChu:LabNotesMDA/2014-2-10

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Use of Qiagen Protease and Gibco Trypsin in Real Time MDA

  • Tried Roche cOmplete protease inhibitor on both Protease and Trypsin. 1.5ml water was added to one mini tablet to make 7x inhibitor cocktail.
  • Protease was diluted in water to 24mAU.
  • Trypsin was diluted to 0.5x in water.
  • 1000 PGP1 cells in 10ul were used. 10ul 400mM KOH denaturation buffer was added and incubated for 3min in room temperature. Then 10ul 400um HCl neutralization buffer was added and transferred to ice.
  1. Heat deactivating protease: 3ul of the above (~100 cell lysate) was added to 1ul protease and then transferred to 45C for 15mins and 75C for 15mins. 1ul water was added. (5ul volume)
  2. Inhibitor for protease: 3ul of the above (~100 cell lysate) was added to 1ul protease and then transferred to 45C for 15mins. 1ul inhibitor was added. (5ul volume)