Preparing Samples in PAGE Gels for LSC

Complex radioactive samples are often fractionated on polyacrylamide gels. Analysis of radiolabeled samples in electrophoretic gels follow the same pattern as that on TLC plates. The gel is analyzed as a whole for radioactive bands, which are then excised and counted to obtain quantitative results. National Diagnostics' Autofluor can be used to enhance the autoradiography of PAGE Gels. Once bands are located and excised, they can be dissolved using hydrogen peroxide and then counted efficiently.

Counting Samples in Polyacrylamide Gels


AUTOFLUOR FLUOROGRAPHY OF ELECTROPHORESIS GELS

  1. Stain and fix gel as usual.
  2. Rinse gel for 15 minutes in deionized water to remove fixative.
  3. Immerse the gel in Autofluor. Agitate gently for 30 minutes per mm of gel thickness. Pour off the Autofluor and retain for future use. LABEL AS RADIOACTIVE MATERIAL!
  4. Place gel directly onto filter paper and dry under heat (80°C) and vacuum.
  5. Expose at -80°C. 24 hours is generally sufficient for 14C or 3H samples, although up to 72 hours may be required for maximum detection of 3H.

DISOLUTION AND COUNTING OF GEL SLICES

  1. Using the autoradiography film as a template, cut out the band(s) of interest.
  2. Add 0.5 ml 30% H2O2 for every 100mg of gel
  3. Incubate in a LSC vial at 50°C until digested (1-4 hours).
  4. Heat at 37°C for one additional hour to drive off residual O2.
  5. Cool, add 10ml of a scintillation cocktail capable of holding 0.6ml of aqueous material (such as Ecoscint H) and count.


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