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Published July 1986 | public
Journal Article

Enhancement of immunoblot sensitivity by heating of hydrated filters


Immunoblots of either dot or Western type were exposed to heat before reaction with antibody. Dramatic increases in immunoblot sensitivity were seen for certain antigen-antibody pairs after heating of either dry or hydrated nitrocellulose filters at or above 100°C. Heating of filters in the hydrated state improved the linearity of immunodetection and produced the highest signal-to-noise ratio. This treatment greatly increased immunoblot sensitivity with several peptide-generated antibodies, whereas decreased sensitivity was seen with antibodies against native proteins. Heating of hydrated filters after antigen immobilization is thus a potentially powerful way to increase the sensitivity of immunoblot analysis for antibodies that preferentially recognize epitopes in denatured proteins.

Additional Information

© 1986 Academic Press. Received January 17, 1986. We are indebted to Caroline Ray for her expert technical assistance and to Aaron Ciechanover (Technion. Haifa. Israel) for his help m preparing the anti-ubiquitin antibody. We also thank Mark Solomon for comments on the manuscript and Barbara Doran for secretarial assistance. This work was supported by grants from the National Institutes of Health IO A.V. (GM31530 and GM33401) and to P.S.S. (HL01484). P.S.S. was supported by a Damon Runyon-Walter Winchell postdoctoral fellowship.

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