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Run SDS-polyacrylamide gels.
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Prepare blotting transfer buffer: (make 6 liters in dd-H2O)
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Cut a piece of 0.2 µm nitrocellulose to fit gel, and cut pieces of 3mm gel blot filter paper.
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Separate gel plates and remove stacking gel.
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For the overnight transfer apparatus prepare the transfer sandwich as:
(-) Plastic holder padding, wetted in transfer buffer, 2 layers of wet 3 mm filter paper, gel nitrocellulose filter (pre-wet in transfer buffer), then 2 layers of wet 3mm filter paper padding wetted in transfer buffer (+) plastic holder.
NOTE: It is important to remove all air bubbles at each stage of the set-up.
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Lock plastic supports (on the holders) together
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Insert sandwich into transfer tank filled with transfer buffer.
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Place in cold room on stirrer.
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Connect power supply, make sure the (-) electrode is on side of the (-) plastic holder of the sandwich.
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Run at 25 mA equivalent to 45V, overnight or run at 100V for 4h.
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Disassemble sandwich. Stain the nitrocellulose with Ponceau S for ~5 min. and destain in water until bands can be seen.
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Can cut off markers at this stage, and mark filter. We can also freeze the membrane at -20oC at this point.
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Soak the membrane in ~25ml of Buffer A for 20-30 min on shaker
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Soak membrane in ~25ml of Buffer B for 20-30min on shaker.
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Incubate membrane in 20ml of Buffer B + primary antibody for 90 min to overnight shaking in a sealed bag or plastic tub.
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Wash 4X 15min with 50ml of Buffer C.
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Insert membrane in seal-a-meal bag or plastic tub, containing 10-50ml of Buffer B. For seal-a- meal bag make sure no bubbles in sealed bag. Add 0.5 µ Ci/ml of 125 I-Protein A. Incubate for 1-2h.
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Pour off supernatant into radioactive waste. Wash 4X 15min with 50ml of Buffer C, and dispose of supernatant properly.
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Place membrane on filter paper to dry. Wrap in Saran wrap. Expose filter at -70oC overnight or appropriate length of time.