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Protein Extraction from Nicotiana benthamiana Leaf

Instrument: Precellys® 24

Sample: Nicotiana benthamiana leaf

Protocol:

  • Add 0.1 g of N. benthamiana leaf to the 2 ml soft tissue homogenizing kit.
  • Add 750 µl of extraction buffer* (0.1 M Tris-HCl, pH 8.3, 5 mM dithiothreitol, 5 mM EDTA and protease inhibitor).
  • Homogenize in the Precellys at 5500 rpm, 1 x 20 sec run.
  • Incubate plant extract at 4°C for 1 hour.
  • Denature the protein by incubating at 95°C for 10 minutes.

*You may substitute another buffer if your downstream application is different.

Results:

Proteins were analyzed by SDS-PAGE and western blot.  Three GFP-fusion proteins are measured in the gel image below.

Western-blot analysis of protein extracts from N. benthamiana leaf cells

Western-blot analysis of protein extracts from N. benthamiana leaf cells transiently expressing CLV1-GFP, CLV2-GFP, or CRN-GFP.  An anti-GFP antibody was used for detection. The Ponceau stained protein bands of Rubisco are shown as a loading control. WT = wild type.