Biomedical Engineering Reference
In-Depth Information
acrylamide concentration, supplement the last detaining solution
with 1% glycerol.
Agarose gels and polyacrylamid gels with %T
<
8 are dried
on air without vacuum. The gel is wrapped in cellophane and the
envelope is fixed in a frame. The drying needs up to several days.
Agarose gels with less than 1% agarose on a support (glass
plate or GelFix foil) dry on air without any manipulations (cf.
Protocol 4.8.4).
2.7 Autoradiography of Radioactive Labeled
Compounds in Gels
Important!
Pay attention to the local and state rules governing the
use of radioactive materials!
Autoradiography is the method of choice for detection and quan-
tification of radioactive labeled samples in electrophoresis gels,
blotting, or hybridization filters using X-ray films. The main draw-
back of X-ray films is a limited linear range of image density with
respect to the amount of radioactivity.
Whereas samples containing strong radiation emitters as
32
P
or
125
I are subjected to autoradiography directly,
3
H,
14
C, or
35
S
labeled probes need an enhancement by solid-state scintillation.
Mostly dried gels or membranes are used in autoradiography.
If wet gels have to be exposed to films, wrap the gel into an imper-
meable thin foil to prevent chemical development of the film.
Sensitivity of X-ray films can be increased by use of an inten-
sifying screen or by pre-flashing. The following protocol describes
autoradiography using intensifying screen and solid scintillation,
respectively.
Autoradiography in the Presence of an Intensifying Screen
Open an X-ray film cassette in a dark room illuminated with a weak
green or red light. Mount the intensifying screen, place a piece
ofX-rayfilmonit,andlaythedrygelormembraneontop.If
no radioactive ink or radioactive labeled markers are used, prick
a needle through gel and film to allow identification of the exact
position of the gel after development.
Close the cassette and expose at −70
◦
C for several minutes
to hours. Since the time of exposure depends on the amount of
radioactivity, this time has to be proved empirically.
After exposure, open the cassette and develop the film according
to manufacturer's instructions in the dark room. After fixing, wash
the film for 15 min in running water and dry on air.
Pre-flashing increases the sensitivity of X-ray films. Pre-flash
needs some experience to catch the right conditions for high sen-
sitivity and low background.
