Parashar Lab

Thrombin cleavage test

(for Factor X do the same but use Factor X buffer for dilutions, less than 100 mM NaCl is ideal for Factor X)

make 1:10 dilution series of thrombin to 1:100,000 using Tris pH 7.5, 150 mM NaCl (or whatever buffer #1 is)

aliquot 50 μl of 50% resin into 7 tubes

(1) 50 μl ~50% resin + 10 μl buffer #1

(2) 50 μl ~50% resin + 10 μl thrombin

(3) 50 μl ~50% resin + 10 μl 1:10 thrombin

(4) 50 μl ~50% resin + 10 μl 1:100 thrombin

(5) 50 μl ~50% resin + 10 μl 1:1,000 thrombin

(6) 50 μl ~50% resin + 10 μl 1:10,000 thrombin

(7) 50 μl ~50% resin + 10 μl 1:100,000 thrombin

  

incubate a Room Temp for 1 hr

add 35 μl 2X SB to each tube

boil 2’

spin 1 min./14,000 RPM

load 10 μl sup to gel (include a MW marker lane)