View source for Western Blot: probing of the membrane

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!Materials
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|Staining Solution
|250 mL isopropanol; 100 mL acetic acid; 650 mL H<sub>2</sub>O; 0.03% Coomassie
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|Destaining Solution
|10% (v/v) isopropanol, 10% (v/v) acetic acid
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|Blocking Solution
|TBS-T; 2% BSA; 0.05% NaN<sub>3</sub>
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|TBS-Tween
|200 mL 10x TBS; 10 mL Tween; ad 2 L H<sub>2</sub>O
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|ECL-Solution
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|H<sub>2</sub>O<sub>2</sub> (30%)
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=== Procedure ===
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* Upon completed transfer of the proteins to the PVDF membrane, the blotting apparatus is disassembled and the membrane is incubated for ~15 minutes at RT in staining solution with shaking
* Subsequently, the membrane is destained until the marker bands are visible and the background appears white
* The Protein Size Marker bands are marked on the membrane
* The membrane is quickly washed with TBS-T and then incubated in Blocking buffer (Blotto) for 1 h at RT or overnight at 4°C on a shaking platform
* The first antibody diluted in blocking buffer (1:500 – 1:5000) is added (usually 5 – 10 ml of antibody solution are sufficient) and the membrane is incubated overnight at 4°C on a shaking platform (cold room)
* The membrane is returned to RT, the solution containing the 1st antibody is saved in a 15 ml Falcon tube for reuse, and the membrane is washed three times for 10 – 20 min with ~50 ml TBS-Tween
* The membrane is incubated for 1 - 2 h with the HRP-conjugated secondary reagent (1:3,000 – 1:10,000 in 10 ml TBS-T) at RT on a shaking platform
* The membrane is washed again three times for 10 – 20 min each with ~50 ml TBS-T
* Add 10 ml of ECL-solution and 3 μL of H<sub>2</sub>O<sub>2</sub> (30%) to the membrane and incubate about 2 min under shaking
* Place the ECL-soaked membrane on the ECL imager plate and record the luminescence. Transfer the image files in your directory on the central file server