| Contributor: |
The Laboratory of David Drubin at the University of California, Berkeley
|
| |
1. Dissolve 400 mg DNase I in 20 ml cold Coupling Buffer. Dialyze overnight against 1 liter cold Coupling Buffer with 1 mM PMSF at 4°C. Use dialysis tubing with a molecular weight cutoff of 35,000 Da.
2. Remove dialyzed DNase I to a new 50 ml conical tube and reserve 30 μl to assay coupling efficiency later. The DNase I should be yellowish and the recovered volume should be the same as before the dialysis. Keep the DNase I on ice.
3. Resuspend one 25 ml bottle of Affigel-10 by gentle shaking. Wash the Affigel matrix five times with 30 ml of cold ddH2O without letting the matrix get dry. To do this, pour the Affigel into a Kimax 60 ml filter funnel and gently apply the vacuum to the sidearm until the top of the matrix is just barely exposed (do not let the matrix dry out!). Add the water and mix the matrix with a spatula (mix the matrix after adding each wash) and again apply the vacuum until the matrix is just barely exposed. Once the isopropanol is washed out, the matrix is active and should be washed and mixed with the DNase I as quickly as possible.
4. Wash the matrix as before using 30 ml of cold Coupling Buffer. Quickly scoop the gel into the conical tube containing the DNase I.
5. Rotate the mixture at 4°C for at least 4 hr.
6. Rinse a 50 ml capacity Biorad column with coupling buffer. Pour the DNase I/Affigel slurry into the column rinse the conical tube with 25 ml of Coupling Buffer and add it to the column as well. Allow the slurry to settle in the column for at least 40 min.
7. Run 200 ml of Coupling Buffer through the column at 2 ml/min. Collect the first 1 ml and compare its DNase I concentration to the original DNase I concentration to assess coupling efficiency.
8. Run 250 ml G-buffer Block through the column at 2 ml/min. This serves to block unbound Affigel and equilibrate the column with G-buffer. If the column is going to sit for more than a few hours, store it without DTT.
|
| G-buffer |
| 0.2 mM DTT (added just before use)
0.5 mM ATP
10 mM Tris-HCl, pH 7.5
0.1 CaCl2
|
 |
| Coupling Buffer |
| 1 mM PMSF (Caution See Hint 1)
0.1M HEPES, pH 7.2 with KOH
80 mM CaCl2
|
|
|
| |
HEPES
DTT
Potassium Hydroxide
Calcium Chloride
PMSF
ATP
Affigel-10
Tris-HCl
Potassium hydroxide
|
| |
1.CAUTION! This substance is a biohazard. Consult this agent's MSDS for proper handling instructions.
|
