Sample Preparation

Generally, a sample that is prepared for autoradiography should be thoroughly dried to minimize attenuation of the beta emission. The procedure of placing a wet blot or sample in plastic wrap or a baggie seriously degrades sensitivity and resolution, particularly when using isotopes of lower energy (e.g., 35 S), and will totally eliminate effective 3 H detection. Further, it is important that any blotting material used in an experiment be that which has the maximum affinity for the blotted biomolecule; e.g., positively-charged nylon for a nucleic acid. Proper choice of the membrane assures that radioactivity is closest to the surface (defining an active side of the blot) which can be placed closest to film and phosphor to maximize sensitivity and spatial resolution.

The assurance that a blot be capable of stripping and re-probing after drying for autoradiography is of some concern. Generally, if the blotted molecules are appropriately cross-linked to the membrane, and the blot is appropriately blocked and probed, stripping and re-probing after drying is not adversely affected.

The simple operational definition of air-dried is sufficient for use with the BioMax TranScreen, whereas the slightest dampness can cause a severe sticking and/or static problems if a blot is exposed to the surface of a film. The BioMax TranScreen surface effectively resists the adverse effect that water and/or ice would ordinarily have on the phosphor.

Darkroom and Safelights

IMPORTANT: BioMax MS film is extremely sensitive to green, yellow, and blue light and should only be exposed to the red GBX-2 safelight filter, 7.5 or 15 watt bulb only. Use of other safelight filters or higher power bulbs will result in significantly increased background fog on the exposed film. Please refer to instructions and precautions provided with the film. The BioMax TranScreen is robust to light exposure over indefinite periods of time. In addition, the time period of light emission after phosphor exposure is sufficiently short (seconds) as to warrant no special precautions involving dark adaptation of the screen.

Instructions for the use of the BioMax TranScreen System

STEP 1. Under proper darkroom conditions, remove the film from the box and insert it into the BioMax TranScreen folder. The BioMax TranScreen folder is designed to help minimize film fogging due to poor darkroom conditions. Refer to darkroom and safelight section for information on proper darkroom conditions.

STEP 2. Place the active side of the sample onto the phosphor surface of the folder as shown in the diagram.

STEP 3. Place the film/screen combination into a cassette and close the cassette.

STEP 4. The cassette should then be placed into a freezer (-60 to -80C). It is important to use the freezer to maximize the sensitivity of the system.

STEP 5. After exposure, allow the cassette to warm to room temperature for at least 15 minutes prior to opening. This will help prevent damaging the cassette hinge and minimize water condensation forming on the exposed film, sample and BioMax TranScreen screen.

STEP 6. Under proper darkroom conditions, open the cassette and disassemble the constructed sandwich. The BioMax TranScreen folder is notched in one corner to facilitate easier access to the film.