Post Electrophoretic Analysis
Autoradiographic Enhancement with Autofluor
Autoradiographic Enhancement with Autofluor: The Procedure
National Diagnostics’ Autofluor is an extremely sensitive, water based fluorographic enhancer for autoradiography on gels, TLC plates or paper chromatograms.
- After staining, fix the gel with 5% glacial acetic acid, 5% isopropyl alcohol, and 90% water. Fix for 15 to 20 minutes. Pour off fixing solution and discard according to radioactive disposal procedures.
- Rinse the gel in a continuous flow of water for 15 minutes to ensure the complete removal of any acetic acid residue. To prevent crystal formation, it is important that the gel be thoroughly rinsed after fixing. Should the gel develop white crystals on contact with Autofluor, dissolve the precipitate by soaking the gel in a solution of 1g sodium carbonate/100ml water or in tris buffer. Soak the gel in Autofluor until the white precipitate is fully dissolved. Repeat from the beginning of Step 2.
- Cover gel with Autofluor until the depth of Autofluor is twice the thickness of the gel. Gently agitate in Autofluor for 30 min/mm of gel thickness. Pour off remaining Autofluor and retain for future use. Label reserved material as radioactive. Autofluor may be reused several times before a diminishing response is observed.
- Place directly on filter paper and dry on gel dryer under heat (80°C) and vacuum. DO NOT WASH GEL.
- The gel will have a white to light tan sparkling appearance similar to freshly fallen snow.
- Place on film and expose at -70°C. Due to the higher light output of the Autofluor phosphor, less exposure time is needed for gels treated with Autofluor than for gels treated with PPO/DMSO. Sufficient exposure time for a 5000 dpm/band is 24 hours. Overexposure of the film will cause the bands to become fuzzy and resolution to be lost.
- Develop film according to manufacturer’s instructions.
PAPER CHROMATOGRAPHY AND TLC PLATES
- Spray twice or dip plates in Autofluor and allow to dry.
- Place on film and expose at -70°C.
TO MAXIMIZE AUTOFLUOR EFFICIENCY
- If gels crack or stick during drying, add 0.5% (5ml/liter) of glycerol directly to the Autofluor before using.
- Since Autofluor is inducted into the gel by crystallization in situ as opposed to precipitated, it is advantageous to form the smallest crystals possible. This is accomplished by drying as quickly as possible under the strongest vacuum possible. A vacuum pump with a good seal on the dryer is preferred over a “house vacuum.” After the gel appears dry, turn off heat and continue vacuum for another 1/2 hour.
NEXT TOPIC: Protein Visualization – Fixing Proteins on Gels
- UV Shadowing
- Uneven Staining
- Staining Proteins Immobilized on Membranes
- Staining Protein Gels with Coomassie Blue
- Southern Blotting
- Smeared Bands
- Silver Staining Protein Gels
- Silver Staining DNA Gels
- Protein Fixation on Gels
- Post-Electrophoretic Visualization with Nuclistain
- Overview of Western Blotting
- Northern Blotting
- Method for Western Blotting
- Mechanism of Immunostaining
- Mechanism of Immunostaining
- Immunostaining with Alkaline Phosphatase
- Guide Strip Technique
- Faint bands, low background
- Faint Bands, High Background
- Ethidium Bromide Staining
- Enzyme Linked Immunosorbent Assay (ELISA)
- Coomassie Blue Stain- Troubleshooting
- Blotches on Gel
- Autoradiographic Enhancement with Autofluor
- An Overview of Northern and Southern Blotting
- Alkaline Blotting
- Sample Preparation
- Electrophoresis Buffers
- Blotting and Detection Buffers
- Gel Matrices
- Gel Accessories
- Ultra Pure Reagents