Protein extraction experiments on woody plants
Within this chapter, we present a protocol for extracting total protein from wood-forming tissues (secondary xylem in differentiation). This protocol has been used on a range of plant organs (roots, leaves, pollen, buds, flowers, lamina, and phloem) from both broadleaf (oak and aspen) and coniferous (pine) plants. The source for this experiment is the "Guide to Plant Proteomics Experiments" [France] H. Tillmant, M. Zivi, C. Damerweil, V. Mitschine, eds.
Operation method
Protein extraction from woody plants
Materials and Instruments
Bark Move 3.1 Extraction of total protein (see Note 5) For more product details, please visit Aladdin Scientific website.
Precipitation buffer Rinsing buffer Dissolving buffer
Centrifuge tube
( 1 ) Empty, uncorked 10 ml centrifuge tubes are weighed. Flexible polycarbonate high speed centrifuge tubes with screw caps and rounded bottoms are used.
( 2 ) Cell fragmentation: 500 mg of fresh tissue (stored at -80°C) is powdered in liquid nitrogen using a mortar and pestle.
( 3 ) Re-dissolve the powder with 8 ml of pre-cooled Precipitation Buffer.
( 4 ) Transfer the mixture to a weighed centrifuge tube.
( 5 ) Rinse the mortar with 2 ml of Pre-cooled Precipitation Buffer.
( 6 ) Transfer the rinse solution to a centrifuge tube as well.
( 7 ) Gently turn the tube up and down for 15s to mix, then allow the protein to settle at -20°C for 1h.
( 8 ) Centrifuge at 16000 g for 15 min (see Note 6).
( 9 ) Gently pour out the supernatant from the centrifuge tube.
(10) Rinse the sediment in the centrifuge tube with 10 ml of pre-cooled rinse buffer (to remove residual TCA) and allow the tube to react at -20°C for 1h.
(11) Centrifuge at 16000 g for 10 min (see Note 6).
(12) Vacuum dry the precipitate for 2 h.
(13) Break the precipitate into powder with a glass rod.
(14) Weigh the centrifuge tube with the dried precipitate.
3.2 Dissolving proteins
( 1 ) Resuspend the powder with dissolution buffer. Dissolve each milligram of powder in 10-30 μl of buffer. This buffer contains a mixture of dissociatives (urea, thiourea), detergent (Triton-X 100, CHAPS), reducing agent (DTT), and carrier amphoteric electrolyte (IPG buffer).
( 2 ) Remove insoluble material (e.g., cellular debris) by centrifugation at 400 g for 4 min at room temperature.
( 3 ) Pour the supernatant into a clean centrifuge tube.
( 4 ) Centrifuge at 400 g for 4 min at room temperature and pour the supernatant into a clean centrifuge tube.
( 5 ) Store the supernatant at -80°C.
( 6 ) Concentrated proteins can be quantified in more than 6 replicates according to the method described by Ramagli et al [8]. Calculate the average concentration and sample 300 μg of protein per IPG strip.