Transfer Membranes

Washes
When the hybridization is complete, wash the membrane in small trays in the following wash solutions:
2X SSC, 0.1 % SDS 6 minutes with one exchange at 3 minutes at room temperature
0.2X SSC, 0.1 % SDS 6 minutes with one exchange at 3 minutes at room temperature
0.16X SSC, 0.1 % SDS 30 minutes with one exchange at 15 minutes at room temperature
05
protocol
Post Hybridization
Block the filter by rinsing for one minute in Buffer 1, followed by an incubation step in 3 % Fraction V BSA in freshly made
Buffer 1 for one hour at 65°C.
Buffer 1 Buffer 2
2 mM Magnesium Chloride 0.1 M Tris · HCl (pH 9.5)
0.1 M Tris · HCl (pH 7.5) 0.1 M NaCl
0.1 M NaCl 50 mM Magnesium Chloride
0.05 % Triton-X 100
Chromogenic Detection
Expose the blot to 7.0 ml of streptavidin detection solution (prepare using 1.0 µl streptavidin-alkaline phosphatase stock
solution per 1.0 ml of Buffer 1) per 100 cm 2 of filter area for 5 minutes/side with gentle mixing. Wash the membrane in
Buffer 1 for 30 minutes (with one exchange at 15 minutes) and Buffer 2 for 10 minutes.
Visualization
For each 100 cm 2 of membrane, prepare a solution containing 33 µl of Nitro Blue Tetrazolium (75 mg/ml in 70 %
dimethylformamide) and 25 µl of 5-bromo-4-chloro-3-indolyl-phosphate (BCIP; 50 mg/ml in dimethylformamide) in 7.5
ml of the incubation mixture referenced above (Buffer 1 with 3 % Fraction V BSA). Place the membrane and solution in a
heat-sealable bag in the dark. Color development should occur within 30 minutes.
Post Detection
Stop the reaction by allowing the membrane to soak for 1 – 2 minutes in a solution of 20 mM Tris (pH 7.5), 0.5 mM
disodium EDTA, followed by baking for 1 – 2 minutes at 80°C.
Alkaline Phosphatase Buffer Solution
Horseradish Peroxidase Substrate Buffer Solution
100 mM Tris · HCl (pH 9.1) 2 mg/ml Tetramethyl Benzidene in
100 mM NaCl 00 % Ethanol and H 2O 2 at 0.0014 %
5 mM MgCl 2 0.01 M Sodium Citrate
5 mM Nitro Blue Tetrazolium 0.01 M EDTA (pH 5.0)
3 mM 5-bromo-4-chloro-3-indolyl-phosphate (BCIP)
After membranes are adequately stained, rinse briefly in deionized water. Dry the membranes between filter paper, or bake
in a vacuum oven.
t o c o l s
© 2008 AppliChem • Transfer Membranes 27