Summary

Autoradiography is an image formed on photographic film by radiation from a radioactive substance. The radioactive substance could be from a radiometric assay or from metabolic labelling of tissue. In the case of biochemical assays, the labelled products are separated from the radioactive substrates either by extraction or separation using chromatographic techniques, commonly thin layer chromatography (TLC; see also Sec. C.2). The TLC plates can then be directly exposed to the photographic plates. In the case of labelled tissues, the compound of interest that could be a metabolite or nucleic acid is radiolabelled and incubated with the tissue. The tissue is washed and then exposed to the photographic plates. The localisation of the radioactive spot indicates the localisation of the molecules of interest. In specialised cases such as Western blots or Southern blots, the samples are probed with a radioactive probe or enzyme that induces the formation of photons. This will again darken photographic films. 35S, 14C, 32P and 125I can be visualised by autora-diography, while 3H requires enhancer materials due to its low energy of emission. In addition, exposure towards the film can be enhanced by the use of screens that reflect the radiation or photons.

Protocol 1:

(1) Dry the tissue slide/gel/TLC plates.

(2) Label them with the isotope or the photomarkers to know the orientation.

(3) Transfer the slide/gel/TLC plates to a transparent plastic bag.

(4) Align them on the cassette containing the screens.

(5) In the dark room under red light, open the film pack and keep it carefully on the top of the plate.

(6) After a specified time point, remove the film and develop it.

(7) If the signals are too low, keep the plate for a longer time.

106 ♦ Autoradiography

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Suggested Readings

1. Abelson JN and Simon MI (1990) Guide to Protein Purification, Vol. 182, Academic Press.

2. Abelson JN and Simon MI (1991) Protein Phosphorylation, Part A: Protein Kinases: Assays, Purification, Antibodies, Functional Analysis, Cloning, and Expression, Vol. 200, 1st ed., Part A (Methods in Enzymology), Academic Press.

3. Ahmed H (2004) Principles and Reactions of Protein Extraction, Purification, and Characterisation, CRC.

4. Bonifacino JS, Dasso M, Harford JB, Lippincott-Schwartz J and Yamada KM (2004) Short Protocols in Cell Biology, John Wiley & Sons.

5. Eisenthal R and Danson M (2002) Enzyme Assays: A Practical Approach (The Practical Approach Series), 2nd ed., Oxford University Press, USA.

6. Freshney RI (2005) Culture of Animal Cells: A Manual of Basic Technique, 5th ed., Wiley-Liss.

7. Fried B (1999) Thin-Layer Chromatography, Chromato-graphic Science, 4th ed., CRC.

8. Gore MG (2000) Spectrophotometry and Spectrofluorimetry: A Practical Approach, 2nd ed., Oxford University Press, USA.

9. Gunstone FD (1996) Fatty Acid and Lipid Chemistry, 1st ed., Springer.

10. Gurr MI, Harwood JL and Frayn KN (2002) Lipid Biochemistry, 5th ed., Iowa State Press.

11. Hames BD (1998) Gel Electrophoresis of Proteins: A Practical Approach, 3rd ed., Oxford University Press, USA.

12. Harrison MA, Rae IF and Harris A (1997) General Techniques of Cell Culture Handbooks in Practical Animal Cell Biology, Cambridge University Press.

13. Hibbs AR (2004) Confocal Microscopy for Biologists, 1st ed., Springer.

14. Nelson DL and Cox MM, Lehninger Principles of Biochemistry, 4th ed.

15. Reith AD (2001) Protein Kinase Protocols (Methods in Molecular Biology), 1st ed., Humana Press.

16. Roe S (2001) Protein Purification Techniques: A Practical Approach, 2nd ed., Oxford University Press, USA.

17. Roskams J and Rodgers L (2002) Lab Ref: A Handbook of Recipes, Reagents, and Other Reference Tools for Use at the Bench, Cold Spring Harbor Laboratory Press.

18. Schantz JT and Ng KW (2004) A Manual for Primary Human Cell Culture, World Scientific Publishing Company.

19. Schwartz LM and AshwellJD (2001) Methods in CellBiology, Vol. 66: Apoptosis, Academic Press.

20. Spector DL and Goldman RD (2005) Basic Methods in Microscopy: Protocols and Concepts from Cells, a Laboratory Manual, Cold Spring Harbor Laboratory Press.

21. Voet D and Voet JG (1995) Biochemistry, 2nd ed., Wiley.

22. Yin XM and Dong Z (2003) Essentials of Apoptosis: A Guide for Basic and Clinical Research, Humana Press.

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Abbreviations

APS

ammonium persulfate

ATP

adenosine trisphosphate

BCIP

5-Bromo-4-chloro-3-indolyl phosphate

BSA

bovine serum albumin

CHO

Chinese hamster ovary

CM

carboxymethylcellulose

DAB

3,3-diaminobenzidine

DEAE

diethylaminoethyl

DMSO

dimethyl sulfoxide

DTT

dithiothreitol

EDTA

ethylene diamine tetraacetic acid

EGTA

ethylenebis(oxyethylenenitrilo) tetraacetic acid

FBS

fetal bovine serum

GST

glutathione transferase

HEPES

4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid

HRP

horseradish peroxidase

IgG

immunoglobulin G

NBT

nitroblue tetrazolium

NTA

nitrilotriacetic acid

O.D.

optical density

PAGE

polyacrylamide gel electrophoresis

PBS

phosphate buffered saline

PI

propidium iodide

PMS

phenazine methosulfate

pNA

p-nitrophenolate

pNPP

p-nitro phenyl phosphate

PS

penicillin/streptomycin

PtdIns

phosphatidylinositol

RIPA

radio-immunoprecipitation assay buffer

RPMI

Roswell Park Memorial Institute

RPMI

revolutions per minute

RT

room temperature

SDS

sodium dodecyl sulfate

TBST

Tris buffered saline Tween 20

TCA

trichloroacetic acid

TdT

terminal transferase

TEMED

tetramethylethylenediamine

TLC

thin layer chromatography

Tris

Tris (hydroxymethyl)-aminomethane

TUNEL

terminal transferase dUTP nick end labelling

XTT

(2,3-Bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-

5-carboxanilide)

Appendices

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