Bitan:Autoradiography of hot blots

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TBE-Urea Gel Electrophoresis and Autoradiography of the gels

Autoradiography of Hot Membranes

 

1. Prepare the blots by spotting proteins of interest on the membrane.

2. Incubate the membranes with the required amount of labeled RNA, usually 500,000 cpm/ml, for 30 min or 60 min.

3. Clean the surface of the work area with Decon making sure all the contaminating radioactive spots (if present) are removed.

4. Wash the blots under appropriate wash conditions with appropriate wash buffers. Check that the amount of radioactivity in the was solution decreases to background levels after washes before the blot is used for autoradiography.

5. After washes, lay out two layers of plastic Saran wrap. Wrap the membrane in between the two-plied plastic wrap.

6. Expose the membrane wrapped in plastic to a sheet of autoradiography X-ray film inside an exposure cassette in the dark room, under safe light.

7. Appose a Denville intensifying screen on the film such that the film is sandwiched between the intensifying screen and the blot. Use the smooth side of the screen for apposition to film.

8. Leave the cassette at −20 °C for 2–3 days according to the level of radioactivity on the blot after assessing by the Geiger counter. The time has to be optimized by trial and error.

9. Develop the film in the dark room under safe light after 2–3 days using an automatic film developer.

 

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