1. 400 mls induced Ecoli/500ul resin. Estimated 0.5
μg/μl packed,
washed beads (7.5e12 molecules/ul bead).
2. 6 μl of 50% slurry shot into 100-200
μl of
0.1% DM,1 mg/ml BSA,40 mM Hepes-Cl
PH 7.8, 200 mM AS (can substitute 50% AS). Assay is typically
performed at 200 nM 3H-cAMP. 3H-cAMP in the stock bottle is typically 40
mM (about
200-fold). Try range of 2-10000 nM for Scatchard. For
non-specific binding add 100 uM.
3. Incubate for 5 minutes (25 minutes is OK). Spin 2 minutes.
4. Carefully remove supernatant with drawn out pasteur. Alternate is to move the
200 μl sample to 50 μl of 9:5 A200:A20 oil. Aspirate super plus oil.
5. Add 200 μl 1%SDS. Wash. Count.
This page was last edited
06/26/2003