Abstract
Immunoblotting is used to determine many important characteristics of proteins. After electrophoretic separation, proteins are transferred to a membrane and reacted with a specific antibody. The antibody-protein complex is then visualized by radiographic, chromogenic, chemiluminescent, or more recently described fluorescence detection methods. Fluorescence-based detection offers some advantages over other approaches, including increased sensitivity, improved quantifiable range, and the ability to detect multiple antigens on the same blot. However, this technique is unavailable for analysis of green plant tissues by standard extraction methods because of contaminating autofluorescent pigments. We have compared 3 methods for extracting protein from plant tissue for use with infrared fluorescence-based immunoblot analysis. We report a trichloroacetic acid-acetone method that effectively eliminates autofluorescence while retaining the immunogenicity of a target protein.
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Abbreviations
- TBST:
-
tris-buffered saline with 0.1% Tween
- TCA:
-
trichloroacetic acid
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Shultz, R.W., Settlage, S.B., Hanley-Bowdoin, L. et al. A trichloroacetic acid-acetone method greatly reduces infrared autofluorescence of protein extracts from plant tissue. Plant Mol Biol Rep 23, 405–409 (2005). https://doi.org/10.1007/BF02788888
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DOI: https://doi.org/10.1007/BF02788888