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MOLECULAR BIOLOGY: WORKING WITH PROTEINS

PROTEIN DETECTION: GEL ELECTROPHORESIS

AUTORADIOGRAPHY

Autoradiography
 
Procedure
1. Take an autoradiography cassette, a box of film, a timer (if short timed exposures are required), and the dried gel (wrapped in plastic wrap) into the darkroom (See Hint #1)

2. In the darkroom, with the lights off (or only the red safety light on), remove a single piece of film from the box and reclose the box.

3. Place the gel in the cassette with the gel facing up.

4. Place any orientation marks that are desired on the surface of the plastic wrap covering the gel (using a radioactive pen, or light emitting markers or stickers).

5. Place the single sheet of film on the gel (See Hint #2) and carefully close the cassette (See Hint #3).

6. Exposure times (See Hint #4) will vary depending on the isotope, or chemiluminescence being used, as well as the technique being employed. As a rough guide, Northerns and Southerns may take anywhere from 1 day to 2 weeks. Exposures of Westerns (using the ECL method) generally range from 10 seconds to 5 minutes. RNase Protection Analysis exposures usually range from 1 day to 3 days. Most methods of gel analysis by autoradiography require multiple exposures.

Solutions
This bioProtocol does not use any solutions
 
BioReagents and Chemicals
This bioProtocol does not use any reagents
 
Protocol Hints
1. Wet gels should be wrapped thoroughly in plastic wrap to prevent any leakage of fluid onto the film.

2. If an enhancing screen is required, the screen is placed on top of the film, facing the gel. The entire cassette is then closed and placed at -70°C until the desired exposure time is reached.

3. The cassette should only be opened in the dark room prior to film development.

4. The development of film should be performed according to the manufacturer or specific laboratory specifications.