Biomedical Engineering Reference
In-Depth Information
Autoradiography in the Presence of a Scintillator
The radiation of
3
H,
14
Cand
35
S has to be converted into light by
scintillators.
A
methanol:glacial acetic acid:pure water 5:5:5 (v/v/v)
Solutions/Reagents
B
16% sodium salicylate (w/v) in ddH
2
O
radioactive ink: mix some kBq of any
14
C-labeled compound
with a little amount of refill and refresh a pen with it. Handle
this pen with care and avoid contaminations.
27
C
Equilibrate the gel after staining in Soln. A for 20 min.Washthe
gel with ddH
2
O twice in a 20-fold of its volume for 20 min each,
followed by an equilibration in Soln. B for 30 min. Dry the gel and
expose to the X-ray film at −70
◦
C as described above.
Blotting membranes and TCL plates are processed analo-
gously
28
. Incubate the dry plate or membrane in Soln. B for 5 -
10 min, dry again, and expose to film.
Variant
The scintillator 2,5-diphenyl-oxazole (PPO) in DMSO may be used
instead of salicylate:
D
22% PPO (w/v) in DMSO (solution is reusable for several
Solutions/Reagents
times)
Attention!
Wear gloves and avoid skin contact toDMSO-containing
solutions, because DMSO penetrates skin very effective.
Incubate the gel fixed with Soln. A in a 20-fold volume of DMSO
for 30 min. Replace DMSO by the same volume of fresh DMSO and
agitate gently for additional 30 min. The DMSO bathes may be used
several times when stored separately and used in the same order.
Shake the gel in 4 vol.ofSoln.Dfor2-3h and transfer to water
after that time. Let the gel for 1 h in water and then dry.
Expose the dry gel to X-ray film in a cassette with intensifying
screen at −70
◦
C as described above.
Quantification of radioactivity is possible by densitometry
(scanning) of the developed film. It should be taken into considera-
tion that the optical density of the film is not linear proportional to
the amount of radioactivity, especially at lower radioactivity. Dot
defined amounts of radioactivity onto a part of the gel or mem-
brane and use the obtained darkening to construct a calibration
curve.
Another possibility of quantification is to cut the bands of in-
terest and counting in a liquid scintillation counter after mincing
the gel pieces.
27
Instead of radioactive ink, pens with phosphorescent dyes are available.
28
Lucher LA, Lego T (1989) Anal Biochem 178:327
