P. Fajer page 2 of 2
Purification of Skeletal Troponin (Hui)
Day_1:
High Salt Extraction: (50 g ether powder/2L)
Stock / amount needed/L1 M KCl / 2.5 M / 400 ml
0.1 mM CaCl2 / 1 M / 0.1 ml
25 mM Tris-HCl pH=8.0 / 1 M / 25 ml
2 mM DTT / 308.6 mg
1. 20 g ether powder, add 400 ml high salt solution to the Warring blender. (15”on, 15”off)x3. Add 500 ml buffer, blend (15”on, 15”off)x2. Stir at low speed in cold room overnight.
Ether powder is extracted overnight with a 15:1 (v/w) ratio of high salt buffer.
Day_2:
Dialyzing Buffer:
Stock / amount needed/L0.1 mM CaCl2 / 1 M / 0.1 ml
2 mM DTT / 308.6 mg
10 mM Imidazole pH=7.0
0.02 % NaN3
50 mM KCl (Buffer A) / 2.5 M / 20 ml
1 M KCl (Buffer B) / 2.5 M / 400 ml
1. Centrifuge in RC-5C, GSA rotor, 8000 rpm, f or 11 min. Resuspend the pellet in 200 ml high salt extracting solution for 20 min, 4oC. Pellet is resuspended with a 7.5:1 (v/w) ratio of starting tissue.
2. Centrifuge in RC-5C, GSA rotor, 8000 rpm, f or 11 min. (Save the pellet in the cold room.)
3. Combine the supernatant (take 100 ul for gel sample). Vol = ______ml, pH~_____.
4. Lower pH to 4.5 with 1 N HCl to precipitate TM, need 1 N HCl ~ ______ml.
5. Centrifuge in RC-5C, GSA rotor, 8000 rpm, f or 11 min. (Save the pellet as crude TM.) Adjust the pH of the supernatant (take 100 ul for gel sample) to 8.0 with 1N KOH, need ~______ml.
6. Slowly add (NH4)2SO4 powder. (10-40% saturation)
230 g (NH4)2SO4/L of supernatant.
Volume of the sup. ______ml, need (NH4)2SO4 ______g.
pH should be kept between 7 and 8 with 1 N KOH. Need ~______ml.
7. Centrifuge in RC-5C, GSA rotor, 8000 rpm, f or 11 min. Take 100 ul of supernatant for gel sample.
8. Slowly add (NH4)2SO4 powder to the supernatant. (40-60% saturation)
125 g (NH4)2SO4/L of supernatant.
Volume of the sup. ______ml, need (NH4)2SO4 ______g.
pH should be kept between 7 and 8 with 1 N KOH. Need ~______ml.
Stir in cold room at low speed for 1 hr.
9. Centrifuge in RC-5C, GSA rotor, 8000 rpm, f or 11 min.
Resuspend the pellet in a minimum volume 10 mM Imidazole, 50 mM KCl, 0.1 mM CaCl2, 0.1 mM DTT, 0.02% NaN3, pH 7. Dialyze 4 times against 1 liter of the same buffer overnight (pellet from 150 g ether powder).
Day_3:
1. The sample is then loaded onto a 5 x 50 cm Cibacron Blue-Sephacryl column equilibrated with the same buffer; 25-ml fractions are collected. The column is eluted with 1 liter of equilibrating buffer until the OD280 return to baseline. The column is then eluted with column buffer containing increasing concentration of KCl. Are volumes are 1 liter of each for 0.1, 0.2, 0.3, 0.4, 0.5, and 0.6 M KCl steps.
2. For re-use the column is first wash with 1.5 liter of equilibrating buffer that contains 6 M urea until the OD280 return to baseline and then with the column buffer until reequilibrated.
3. Pooling tubes 201-220 yielded 612 mg of pure troponin. Pure troponin is eluted from this column at a conductivity between 10.8 and 15.4 mS. The pure troponin can be stored frozen in solution at -80o or lyophilized and stored at -20o.
Fajerlab under EDITMethods.doc 10/20/1999 4:55 PM10/20/1999 4:55 PM