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Methodology for Amino Acids and Proteins

 
     
 
Fluorography of polyacrylamide gel
 
Fluorography is an improved version of autoradiography but in the presence of a fluorescing compound. It is a technique to determine the radioactivity in gels and other media by a combination of fluorescence and photography. When radioactively labeled macromolecules such as proteins are separated by electrophoresis much of the radiation is absorbed by the gel and the autoradiography of such gel gives poor results. For low-energy emitters such as tritium and 14C the sensitivity of fluorography is many-fold higher than that of autoradiography. The fluorography may be photographed for qualitative information or analyzed by scanning for quantitative results.
 
 


Principle
In fluorographic technique, a fluoro/scintillator impregnated into the gel absorbs the radiation from the isotope and re-emits light that passes through the gel to the film producing a photographic image analogous to an autoradiograph.
 
 
Materials
Dimethyl sulphoxide (DMSO)
PPO (2,5 dipheynl oxazole) Solution: Dissolve 22g PPO and make up to100mL in DMSO
Fixing Solution: 7% acetic acid, 20% methanol in distilled water.
Photographic Flash Unit (with red screen on the flash window)
X-ray Film (Kodak X-omat AR)
Gel Dryer
Film Developer
 
 


Procedure
1.
Fractionate radioactively-labeled polypeptides by SDS-PAGE as described under ‘SDS-PAGE of Proteins’. Stain and destain the gel, if required. Otherwise the proteins are fixed  by immersing the gel in the fixing solution foe an hour.
2.
Immerse the destained/fixed gel in 20 volumes of DMSO for 30min to dehydrate the gel. Repeat this step for complete dehydration.
3.
Immerse the gel in the PPO solution to impregnate PPO into the gel for 3h. the gel is shaken slowly in a large petri-dish throughout the steps1-3. The gel shall appear shrunk due to dehydration.
4.
Transfer the gel gently and evenly to a dish containing a large volume of water for 30-60min. in water, PPO precipitates so that the gel turns opaque. Clean the gel in water.
5.
Dry the gel under vacuum at 70°C on a sheet of 3mm filter paper, carefully. In a photographic dark room, take out an X-ray film and lay a sheet of transparent paper over it. Presensitize the film using a battery-operated small flash unit (camera flash) from 2m height in the dark. Assemble the film and dried gel, with the presensitized side of the film facing the dried gel, between a fold of the thick black sheet. Sandwich this assembly between two plywood plates 3-4 layers of aluminum foil and hold them firm using bulldog clips. A cassette may be used, if available instead of the above assembly.
6.
Place the assembly/cassette at -20°C in a freezer for 2-4 days to expose the film to the gel. In the case of gel which received low amounts of radioactivity, the exposure is done at -70°C to get the film sufficiently exposed.
7.
Take out the assembly/cassette from the freezer after the appropriate exposure time and allow to warm up for 1-1.5h at room temp.
8.
In the dark room, open the assembly and develop the film.
 
 
Notes
1.
DMSO diffuses into the skin very easily proving harmful. Wear disposable gloves while handling.
2.
Labeling of polypeptides is done using radioactive (3H, 14C or 35S) amino acids either in in vivo or in vitro in a cell-free protein-synthesizing system with mRNAs. Use high specific activity precursor. Load each lane of the gel with equal counts of radioactivity.
3.
The times given in step 1-3 are sufficient for 15% acrylamide gel, thickness up to 1.5mm. thicker or more concentrated gel will require longer periods in all solutions.
4.
Drying the gel should be carefully done. A commercially available gel dryer id preferred. Where such facility is unavailable, the procedure described below is useful. Place the PPO impregnated washed, opaque gel into a sheet of 3mm filter paper slightly larger than the gel itself. Do not trap air bubbles between them. Place them, gel uppermost, on top of a porous rigid polyethene pad. Introduce the whole assembly into a thick gauge polythene bag(s). Turn upside down such that the porous pad is on top of the gel inside the polythene bag. Place at the center of the pad a glass funnel steam facing upward. Fasten the mouth of polyethene bag around the funnel steam using elastic bands or thread so that the bag is leak-proof. Connect the steam to a water pump vacuum line. Water in the gel is removed as droplets by suction. Place the gel assembly after 5-10min over a hot plate at 70°C to hasten the drying. Many factors such as the gel thickness, efficiency of vacuum pump, the presence of DMSO in the gel etc., influence the gel drying period. Gel cracking may occur as an acute problem if the vacuum is turned off when the gel is partly dry. Continue until the gel is completely dried which may require 3-4h or even longer. Finally, the dried gel on the filter paper is removed and used for fluorography.
5.
Pre- sensitizing of the film is done to improve the sensitivity and linearity of response of the film. The flash window of the unit should be covered with two layers of red filter (transparent sheets). While pre-sensitizing neither the aluminum foil nor the gel should be at the vicinity of the film. Due not use the first flash after switching on the unit as it shall be stronger and different from other flashes. Pre- sensitizing conditions such as the height of the flash unit from the film, the n8umber of transparent sheets over the film, the red filter over the flash window etc. should be standard to get satisfactory results on the fluorography.
6.
Do not place the gel-film sandwich near any radiation source during exposure as otherwise the fluorography plate will be fogged.
7.
The exact exposure time is dependent on the amount of radioactivity in the gel. The recommend exposure times are of the order of 24h for 1000-10,000dpm 3H and correspondingly longer for lower amounts of radioactivity. Exposure at lower temperatures (say -70°C) is beneficial than at high temperatures.
8.
The unused PPO in the DMSO solution can be recovered and recycled. Mix one volume of PPO solution with 10 volumes of distilled water and stir continuously. The PPO crystallizes out. Filter, collect the crystals and dry at 25°C for 2-3 days. Dissolve the PPO in a minimum volume of ethanol and precipitate, filter and dry again. Finally, dry the PPO in a vacuum oven for a few days and reuse.
9.
View the developed plate for polypeptides which appear as black bands. Photograph the plate and make prints for qualitative information.
10.
Quantitative information on the polypeptides can be obtained by scanning the X-ray plate (see Quantification of proteins in polyacrylamide gels).
 
 
References
1. Bonner, W M and Laskey, R A (1974) Eur J Biochem 46 83.
2. Laskey, R A and Mills, A D (1975) Eur J Biochem 56 335.


 
     
 
 
     




     
 
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