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 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.
