1. Preparing the Gel - Protein Fixation and Staining (Optional)

After isolating proteins in an acrylamide gel by electrophoresis, the proteins may be fixed or stained before enhancement.  To do this, place the gel in a tray containing fixing solution; polyethelene trays are well suited for this purpose.  Then, agitate the solution for 30 to 60 minutes.  Make sure that the volume of fixer is adequate to keep the gel free-floating.  An efficient solution for protein fixation is composed of 10% (v/v) glacial acetic acid and 30% (v/v) methanol.  If time does not permit the completion of the enhancement procedure, gels can be left in the fixer overnight since longer dwell times have not been found to cause any deleterious effects.

Helpful Hints:

• When using ENLIGHTNING for the fluorography of translated proteins, it is essential to remove the free, labeled amino acids from the protein.  This can be done either prior to gel electrophoresis using a method such as dialysis, or after gel electrophoresis by equilibrating the gel in several changes of fixing solution.
• If the gel contains urea, it must be removed prior to treatment by soaking the gel for 30 minutes in an excess of fixing solution.
• TCA is a quencher and should be avoided whenever possible.
• Due to the severe quenching effects of silver, gels stained with silver must be destained prior to enhancement. 

2.  Impregnating With Scintillators

After fixing or staining/destaining the gel, pour off the solution and discard it according to proper radioactive waste disposal procedures.  Then, for polyacrylamide gels, add a volume of ENLIGHTNING equivalent to five times the gel volume.  The size of the gel and tray will further determine the amount of ENLIGHTNING to use.  Note that the gel should be free-floating and should not stick to the bottom of the tray.  Gently agitate for 15 to 30 minutes.  For two percent agarose gels, the agarose must be at least 3 mm thick to retain sufficient scintillator.  Add enought ENLIGHTNING so that the gel is free-floating (approximately five times the gel volume), then gently agitate for five minutes. 

3.  Drying the Gel

Slide a piece of filter paper beneath the gel; then, carefully lift the filter paper out of the tray and place it on the slab gel dryer.  By transferring the gel in this manner, stretching - a common cause of cracking - can be avoided.  Air bubbles, which form between the gel and the rubber cover of the dryer, are another cause of cracking.  To reduce the formation of air bubbles, cover the gel with clear plastic wrap and then with the rigid plastic sheet supplied with the dryer.  The plastic wrap minimizes contamination and the rigid plastic sheet uniformly presses the gel.  Apply vacuum (refer to Helpful Hints below); then, remove any remaining air bubbles by rolling or gently pressing with a brayer.  Dry under vacuum with heat at a temperature below 95 degrees Celcius. 

Helpful Hints:

• A mechanical vacuum pump provides the most efficient drying.  Insufficient or inconsistent vacuum can cause gels to crack; therefore, house vacuum or water aspirator vacuum sources are not recommended.
• To prevent potentially corrosive vapors and moisture from entering the pump, an acid base vapor trap and a cold trap should be used.
• In general, 0.75 mm thick gels require one hour to dry and 1.5 mm thick gels require 1.5 hours.  For thicker gels, or those with higher acrylamide concentrations (>15%), longer times may be necessary.
• Breaking the vacuum on the dryer before the gel is thoroughly dry can cause the gel to crack.
• Failure to use the rigid plastic sheet often results in cracked gels.
• If the agarose gel begins to melt, lower the temperature of the gel dryer or dry the gel at room temperature. 

4.  Exposing to Film

Place the dried gel against a suitable, blue-sensitive X-ray film, such as Kodak X-Omat AR Film.  Then, expose the film in the temperature range of -76 to -80 degrees Celcius, until the desired visualization level is achieved.  The colder the exposure temperature, the greater the sensitivity achieved.

Helpful Hint:

• If re-exposure of the gel is desired at a later time, store the gel at room temperature in the dark.