C.B.S. Scientific HTLE-7002 - Manual (Page 29)

C. B. S. œ Scientific 29 HTLE-7002
3.6 Autoradiography & Fluorography
Before autoradiography or fluorography we mark the edges of the plates with radioactive india
ink. Several landmarks are chosen to help reorient the film, usually the hatchmarks on the sides
to the plates that line up with the sample and dye origins (see figure in appendix). Often a small
piece of tape or sticker with the plate ID number written in radioactive ink is placed in the upper
left corner as a reference.
For P
32
-labeled peptides or amino acids we perform autoradiography using presensitized Kodak
XAR film and an intensifying screen at 70
°C. Place the presensitized (flashed) side of the film
face up and lay it on top of the plate followed by an intensifying screen. Expose to film for 1 to 7
days depending on the number of counts loaded. Exposure to presensitized film for longer than
7 days is not recommended because the background signal increases.
For either C
14
or S
35
-labeled proteins it is usually the best to use fluorescent indicator to detect
signals. Plates can either be dipped in molten 2-methyl naphthalene containing 0.4% PPO or
sprayed with commercial fluors such as Enhance (NEN). Allow the plates to dry before exposing
to film (
∼20min). Lay a piece of presensitized film, flashed side down, on top of the plate and
expose for 1-15 days depending on the number of counts loaded. When not using an intensifying
screen one can expose the film for considerably longer periods of time (up to two months).
3.7 References
1. Aebersold, R.H., J. Leavitt, R.A. Saavedra, L.E. Hood and S.B.H. Kent. (1987) Internal amino acid
sequence analysis of proteins separated by one- or two-dimensional electrophoresis after in situ
protease digestion in nitrocellulose. Proc.Natl.Acad.Sci USA 84: 6970-6974.
2. Contor, L.F. Lamy and R.E. Lecocq. (1987) Use of electroblotting to detect and analyze
phoshotyrosine containing peptides separated by two-gel electrophoresis. Anal. Biochem. 160: 414-
420.
3. Cooper, J.A., B.M. Sefton, and T. Hunter. (1983). Detection and quantitation of phosphotyrosine in
proteins. Methods Enzymol. 99: 387-402
4. Gibson, W. (1974) Polyoma virus proteins: A description of the structural proteins of the viron based
on polyacrylamide gel electrophoresis and peptide analysis. Virology 62: 319-336
5. Kamps, M.A. and B.M. Sefton (1989) Acid and base hydrolysis of phospho-proteins bound to
Immobilon: A rapid technique for the analysis of phoshoamino acids in gel-fractionated proteins.
Anal. Biochem. 176: 22-27