Protein extraction from leaves
Updated 6/26/2014 08:47 AM
Introduction
Materials
- Leaf Extraction Buffer
- 4% SDS
- 20mM Tris Base
- 2X Protease Inhibitor
Procedure
Leaf prep
- Harvest leaves and introduce around 50mg of fresh material into 1.5mL eppendorfs.
- Add 3-4 glass beads to each tube and freeze in liquid nitrogen. Use any homogenisation method of choice here. We usually use a shaking homogeniser with glass beads with two 5 sec pulses.
- Grind the leaves using a homogeniser and place on ice.
- Add 200µL of Extraction buffer for each 50mg sample and pulse grind on the homogeniser again.
- Incubate at RT.
20 m
- Centrifuge at full speed and remove the supernatant avoiding any plant material
5 m
- Run a BCA assay to quantify the protein content.
- For electrophoresis it is ideal to load 50µg of protein with a SDS-free loading buffer.
This protocol is used for crude extraction of proteins from leaves and may need to be tweaked depending on the protein of interest.