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Detection of tritiated(3H) labelled compounds after Thin Layer Chromatography(TLC).

Samples of tritiated(3H) labelled CTP, UTP, GDP, and TTP were separated using thin layer chromatography(TLC). All compounds were spotted onto the TLC plate as a 2m l sample of stock solution of 1m M, and the specific activities of the stock were as follows:

CTP: 1.7kBq; UTP: 2.7kBq; GDP:2.5kBq; and TTP:6.2kBq.

Standard autoradiography was used in an attempt to visualise the spatial distribution of the various tritiated(3H) labelled compounds. The TLC plate was exposed to preflashed X-ray film , in a cassette, which was placed in a -700C freezer . After 8 weeks the film was developed and was found to be blank.

The same TLC plate was then prepared for Enhanced Autoradiography. The EA-Wax was melted into the TLC plate at a temperature of 550C for a period of 5 minutes. After cooling, the plate was again placed in a cassette with preflashed X-ray film, and placed in a -700C freezer. The film was developed after a period of 7 days. The resultant images are shown.

tlc1a.jpg (7497 bytes) tlc1b.jpg (6078 bytes)
TLC plate image after 8 weeks using conventional autoradiography

The same TLC plate as above but after 7 days using Enhanced Autoradiography

The advantages of Enhanced Autoradiography in this experiment are:

  1. The superior detection efficiency of Enhanced Autoradiography over autoradiography is obvious, by visual inspection. The tritiated(3H) labelled compounds are all clearly visible.

  2. An adequate image could well be available in a shorter time.

  3. Autoradiography produced no result after 8 weeks. It could be more than double this period of time before a usable image was produced.

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