Protein Identification: Overlay Procedures

Abstract

The protein blot overlay assay is a powerful technique for identifying proteins transferred to an immobilizing matrix. It is based on the specific binding of labelled ligands with proteins of interest blotted onto the membrane, followed by detection of the complexes formed.

Keywords: protein–ligand interactions; blot overlay assay

Figure 1.

The protein blot overlay assay for a 2D protein gel.

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References

Dejgaard K and Celis JE (1998) Two‐dimensional Northwestern blotting. In: Celis JE, Carter N, Hunter T, Shotton D, Simons K and Small JV (eds) Cell Biology. A Laboratory Handbook, vol. 4, pp. 482–486. San Diego, CA: Academic Press.

Feizi T (1989) Glycoprotein oligosaccharides as recognition structures. Ciba Foundation Symposium 145: 62–74.

Gromov P and Celis JE (1998) Blot overlay assay for the identification of GTP‐binding proteins. In: Celis JE, Carter N, Hunter T, Shotton D, Simons K and Small JV (eds) Cell Biology. A Laboratory Handbook, vol. 4, pp. 454–457. San Diego, CA: Academic Press.

Hausken ZE, Coghlan VM, Shafer Hastings CA, Reimann EM and Scott JD (1994) Type II regulatory subunit (RII) of the cAMP‐dependent protein kinase interaction with A‐kinase anchor requires isoleucine 3 and 5. Journal of Biological Chemistry 269: 24245–24251.

Hausken ZE, Coghlan VM and Scott JD (1998) Overlay, ligand blotting and band‐shift techniques to study kinase anchoring. In: Clegg RA (ed.) Protein Targeting Protocols. Methods in Molecular Biology, vol. 88, pp. 47–64. Totowa, NJ: Humana Press.

Hoffmann HJ, Gromov P and Celis JE (1998) Calcium overlay assay. In: Celis JE, Carter N, Hunter T, Shotton D, Simons K and Small JV (eds) Cell Biology. A Laboratory Handbook, vol. 4, pp. 450–453. San Diego, CA: Academic Press.

Ramos‐Castaneda J, Imbert JL, Barron BL and Ramos C (1997) A 65‐kDa trypsin‐sensible membrane cell protein as a possible receptor for Dengue virus in cultured neuroblastoma cells. Journal of Neurovirology 6: 435–440.

Further Reading

Clegg RA (ed.) (1998) Protein Targeting Protocols. Methods in Molecular Biology, vol. 88. Totowa, NJ: Humana Press.

Celis JE, Carter N, Hunter T, Shotton D, Simons K and Small JV (eds) (1998) Cell Biology. A Laboratory Handbook, vol. 4. San Diego, CA: Academic Press.

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How to Cite close
Gromov, Pavel S, and Celis, Julio E(Apr 2001) Protein Identification: Overlay Procedures. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1038/npg.els.0002689]