Transfer Membranes

Hybridization
Convert the probe to its single stranded form by adding an equal volume of 0.2 N NaOH and incubate at 37°C 1 – 2 hours.
Remove the prehybridization buffer, add the hybridization buffer with the probe, and incubate at 42°C 12 hours or overnight
with gentle agitation.
04
protocol
Hybridization Buffer
45 % deionized formamide
1X Denhardt’s solution
5X SSC
0.1 % SDS
10 % Dextran Sulfate
20 mM Phosphate buffer/pH 7.0
p r o t o c o l s
Washes
When the hybridization is complete, wash the membrane in small trays in the following wash solutions:
0.16X SSC, 0.1 % SDS, 2 times for 3 minutes each at room temperature
0.16X SSC, 0.1 % SDS, 2 times for 3 minutes each at 64°C;
Rinse in 2X SSC, 2 times for 3 minutes each at room temperature.
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. Repeat incubation step with fresh buffer for an additional 30 minutes.
Buffer 1
0.1 M Tris · HCl (pH 7.5)
0.1 M NaCl
2 mM Magnesium Chloride
0.05 % Triton-X 100
Detection
Remove the membrane from the hybridization solution, and wash in TBS buffer with 5 % Tween-20 for 5 minutes. Additional
washes in TBS buffer may be required.
Add the streptavidin-enzyme complex according to manufacturer’s instructions.
Recommended Buffer Solutions
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)
Post Detection
After the membranes are adequately stained, rinse briefly in deionized water. Dry the membranes between filter paper, or
bake in a vacuum oven.
© 2008 AppliChem • Transfer Membranes 25