TITLE: Leaf Protein Extraction for Immunoblot (Soybean, Cowpea, Tobacco)
AUTHORS: Steven Burgess

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protein extraction protocol was developed for analysis of protein abundance in leaf tissue by immunoblot. It was optimized for dicot species including Glycine max.</div><div class = "text-block"><span style = "font-weight:bold;">Quantities</span><span> - 450 µL protein extraction buffer (PEB) per three 13.4 mm diameter leaf disks (size #7 Humboldt Cork Borer)</span></div><div class = "text-block"><span style = "font-weight:bold;">Note</span><span> from Agrisera: Keeping sample volumes in a range of 0.2-0.5 mL has been found to contribute to better extraction results, an upscale in volume is not recommended, if no cork borer available the suggestion is 500 µL PEB per 100 mg of plant tissue. </span></div><div class = "text-block">- Final volume ~400 µL </div><div class = "text-block">- Expected yield: 1.5 - 6 µg / µL </div><div class = "text-block">- Total yield: 6 - 24 mg protein </div><div class = "text-block">- Amount of total soluble protein to load per lane 30 µg  </div><div class = "text-block"><span style = "font-weight:bold;">Note</span><span>: when using the TissueLyser II it is recommended to use 2 mL centrifuge tubes in conjunction with 4mm beads. 1.5mL tubes are narrow at the end and samples will not grind properly if you use the 4mm beads.</span></div></div>

[STEPS]
?. [Buffer Preparation]
Incubate 4x PEB (if stored at 4 oC) at  for  to re-suspended precipitated SDS in buffer.
50 °C
?. [Buffer Preparation]
Make  1x PEB per sample by diluting 4x stock with dH2O.
500 µl
It is advised to make more 1x PEB than necessary to avoid running out.
?. [Tissue Lysis]
Pre-cool components for tissue lysis
?. [Tissue Lysis]
Pre-cool 4mm SPEX stainless steel grinding beads at   for
-80 °C
?. [Tissue Lysis]
Pre-cool the TissueLyser inserts for LN2 for
?. [Tissue Lysis]
Prepare sample tubes for tissue lysis
?. [Tissue Lysis]
Using forceps cooled in LN2, add one SPEX bead per sample tube.
?. [Tissue Lysis]
Insert tubes into pre-cooled TissueLyser II cassettes, ensuring a balanced number of samples between cassettes.
?. [Tissue Lysis]
Grind tissue for   at 20 Hz
Warning: Do not exceed this frequency, higher frequencies increase the number of cases where the steel beads will break the lid of centrifuge tubes resulting in sample loss. If using the SPEX 2150 beads it is necessary to use 2 mL centrifuge tubes to ensure proper grinding, in 1.5 mL tubes the bead will not reach the tapered bottom leaving samples unground
?. [Tissue Lysis]
Remove cassettes from TissueLyser II and submerge them in LN2 to prevent thawing
?. [Tissue Lysis]
Grind tissue for   at 20 Hz
This repeat is to ensure all tissue is correctly grounded. There will be odd instances where leaf tissue has not properly ground for reasons such as samples sticking to the edge of tubes, check for this on removal, it may be necessary to repeat again.
?. [Tissue Lysis]
Remove tubes with forceps cooled in LN2. Samples can be stored at   for several months before processing further.
-80 °C
It is advised not to proceed directly from samples in LN2 to the next step as they take longer to thaw and resuspend in extraction buffer. A period of 30 mins on dry ice can be beneficial.
?. [Dissolve ground powder]
Add  1x PEB to ground powder (3x 1cm leaf disk).
450 µl
?. [Dissolve ground powder]
Vortex immediately (maximum speed) to mix. Approximately
No lumps should be visible at this stage if the sample is ground well.
?. [Dissolve ground powder]
Heat samples at  for   to denature proteins and inactivate proteases.
95 °C
Prolonged heating can cause cleavage of peptide bonds leading to artifacts. Do not delay heating after sample buffer addition as not all proteases are denatured by SDS and partially denatured peptides are sensitive to protease degradation.
?. [Dissolve ground powder]
Vortex heated samples to shear nucleic acids ( ; max speed)
?. [Dissolve ground powder]
Spin samples for   at   to pellet insoluble material
Centrifuge: 10000 34
the pellet should be white/light-grey an intense green color of the pellet can indicate that disruption was not optimal and extraction conditions need to be adjusted (e.g. improved grinding, or adjusting buffer volume)
?. [Dissolve ground powder]
Transfer supernatant to a new 1.5 mL centrifuge tube and transfer to ice or store at  for up to a month
-20 °C
Be careful not carry over debris