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Star Republic: Guide for Biologists

Western --- dot blot

1. Dot blot can be used to estimate protein concentration in crude or partially purified samples.

2. Cut nitrocellulose membrane to the appropriate size to fit in the dot-blot apparatus. Wet the membrane by immersing it in deionized water. For PVDF membrane pre-wet with methanol for seconds and soak in deionized water. Add sample and standard diluted in 100 m l of PBS/0.05% Tween 20 to each well. Draw the liquid through the membrane by applying suction pump.

2a. Alternatively cut nitrocellulose membrane (not PVDV) to the appropriate size. Draw grids with a pencil. Spot 1 m l of the sample and standard in the center of the grid. Let the membrane dry.

You may make serial dilution of the sample and standard.

3. Proceed to Western.