Materials and Reagents

2 ml microcentrifuge tubes (Fisher Scientific, catalog number: 05-408-138 )
Dialysis tubing (12-14 kDa MWCO) (Spectra/Por, Fisher Scientific, catalog number: 08-667B )
25 mm syringe filters (0.45 μm) (Sarstedt, catalog number: 83.1826 )
Harsh Hakea leaves or roots
Dewar (MVE 20/15) with liquid N₂, and ice bucket
Imidazole (Bioshop, catalog number: IMID508 )
Triton X-100 (Bioshop, catalog number: TRX777 )
Glycerol (Bioshop, catalog number: GLY002 )
Magnesium chloride (Bioshop, catalog number: MAG508 )
Polyethylene glycol 8000 (Bioshop, catalog number: PEG800 )
Phenylmethylsulfonyl fluoride (PMSF, G-Biosciences, catalog number: 786-0555 )
Thiourea (Sigma, catalog number: T-7875 )
Poly (vinylpolypyrrolidone) (PVPP, Sigma-Aldrich, catalog number: P-6755 )
Poly (vinylpyrrolidone) (Bioshop, catalog number: PVP504 )
Intracellular extraction buffer (see Recipes)
Resuspension buffer (see Recipes)
Cell wall extraction buffer (see Recipes)
Dialysis buffer (see Recipes)

Equipment

Zeba^TM spin desalting columns 7K MWCO (0.5 ml) (Fisher Scientific, catalog number: 89882 )
Porcelain mortar (70 mm inner diameter) and pestle
Plastic pellet pestle (Kimble-Chase Kontes, Fisher Scientific, catalog number: K749521-0590 )
Macrosep^® Advance Centrifugal Concentrators (10 kDa MWCO) (Pall Corporation, catalog number: MAP010C37 )
Microcentrifuge (Beckman Coulter, model: microfuge 22R )
PT-3100 Polytron Homogenizer (Kinematica, model: PT-3100)
Magnetic stirrer
4 °C cold room

Procedure

This protocol applies to extraction of intracellular and cell-wall proteins from Harsh Hakea leaves, but can also be applied to roots (e.g., Shane et al., 2014; Shane et al., 2013). For protocols optimized for extracting intracellular and cell wall proteins from leaves, stems etc. of the model plant Arabidopsis thaliana see Shane et al. (2014); Shane et al. (2013) and Robinson et al. (2012).

Extraction of soluble intracellular proteins
1. Quickly freeze freshly harvested tissues in liquid N₂. Store at -80 °C until use.
2. Grind frozen tissues (e.g., ~1 g, comprising a biological replicate) to a powder under liquid N₂ using a pre-chilled mortar and pestle.
3. Transfer an aliquot (e.g., ~200 mg) of frozen tissue powder to a pre-cooled, 2 ml microcentrifuge tube. Do not allow tissue to thaw during this step. Maintain samples in liquid N₂ until all samples are weighed out.
4. Add ice-cold Intracellular extraction buffer (IEB) at 1:4 (w/v) to each sample and vortex until tissue thaws into extraction buffer. Briefly homogenize sample using the Polytron (2 to 4 pulses at high speed for 10 to 15 sec per pulse) while maintaining the microcentrifuge tube on ice. Retain homogenates on ice until all extractions are completed (it is recommended not to exceed 8 to 10 samples per batch to reduce probability of protein proteolysis etc.).
5. Centrifuge homogenates at 17,500 x g for 15 min at 4 °C.
6. Carefully pipette off supernatant (clarified extract) and desalt using a Zeba^TM spin desalting column previously equilibrated in IEB lacking PVPP and PVP. The desalted extract is designated the intracellular fraction of soluble proteins (e.g., ~2-10 mg protein/ml depending upon the tissue type and stage of development).
Washing the insoluble pellets
1. The pellet containing insoluble cell debris is washed twice by resuspending in 1.5-2.0 ml of re-suspension buffer (RB) using a pellet pestle, and centrifugation as above (supernatants are discarded).
2. The pellet is washed as above an additional four times in RB lacking Triton X-100. When completely washed, the pellets will be nearly colourless.
Extraction of cell-wall bound proteins
1. The extraction of cell wall proteins from the washed insoluble pellets is done using a cell wall extraction buffer (CWEB) (see Recipes) (~ 2 ml) while stirring for 30 min at 4 °C.
2. Samples are centrifuged as above and concentrated using the Macrosep^® centrifugal concentrator to ~0.2 ml yielding a protein concentration of ~2 mg/ml [for spectrophotometric determination of protein concentration see Knowles and Plaxton (2013)].
3. Clarify extracts by passing through the 0.45 syringe filter, and then dialyse overnight at 4 °C against 4 L of the dialysis buffer (DB) using Spectra/Por dialysis tubing (12-14 kDa MWCO, 25 mm flat width).
4. The resultant sample contains ‘loosely-bound’ cell-wall proteins.
5. SDS-PAGE of the cell wall versus intracellular fractions followed by immunoblotting using anti-aldolase and anti-phosphoenolpyruvate carboxylase antibodies is performed to confirm absence of immunoreactive -40 kDa aldolase and -100 kDa phosphoenolpyruvate carboxylase polypeptides (i.e., cytoplasmic marker proteins) in the cell wall extracts [for detailed procedure see Shane et al. (2014)].

Recipes

All buffers are made up fresh the day before and stored at 4 °C until used, but no sterilization was done.

Intracellular extraction buffer (IEB, keep on ice)
50 mM imidazole-HCl (pH 7.0)
0.1% (v/v) Triton X-100
10% (v/v) glycerol
10 mM thiourea
2 mM MgCl₂
2% (w/v) polyethylene glycol 8,000
1% (w/v) polyvinyl (polypyrrolidone) (PVPP)
Note: Add fresh to IEB immediately prior to tissue extraction.
1% (w/v) polyvinylpyrrolidone (PVP)
Note: Add fresh to IEB immediately prior to tissue extraction.
1 mM phenylmethylsulphonyl fluoride (PMSF)
Notes:
1. Add fresh to IEB immediately prior to tissue extraction.
2. Prepare a 100 mM stock of PMSF in absolute ethanol and store at -20 °C; PMSF is unstable in aqueous solution. Handle PMSF with care as this protease inhibitor is quite toxic.
Resuspension buffer (RB, keep in ice)
50 mM Tris-HCl (pH 7.4)
10 mM MgCl₂
1% (v/v) Triton X-100
Cell wall extraction buffer (CWEB, keep in ice)
1 M NaCl in 40 mM Tris-HCl (pH 7.4)
10 mM MgCl₂
Dialysis buffer (DB, keep in 4 °C cold room)
40 mM Tris-HCl (pH 7.4)
10 mM MgCl₂

Acknowledgments

This work was supported by the Australian Research Council (grant no. DP1092856 to M. W. S.), as well as grants from the Natural Sciences and Engineering Research Council of Canada and Queen’s Research Chairs program (to W. C. P.).

References

Knowles, V. and Plaxton, W. (2013). Protein extraction, acid phosphatase activity assays, and determination of soluble protein concentration. Bio-protocol 3(17): e889.
Shane, M. W., Fedosejevs, E. T. and Plaxton, W. C. (2013). Reciprocal control of anaplerotic phosphoenolpyruvate carboxylase by in vivo monoubiquitination and phosphorylation in developing proteoid roots of phosphate-deficient harsh hakea. Plant Physiol 161(4): 1634-1644.
Shane, M. W., Stigter, K., Fedosejevs, E. T. and Plaxton, W. C. (2014). Senescence-inducible cell wall and intracellular purple acid phosphatases: implications for phosphorus remobilization in Hakea prostrata (Proteaceae) and Arabidopsis thaliana (Brassicaceae). J Exp Bot 65(20): 6097-6106.
Robinson, W. D., Park, J., Tran, H. T., Del Vecchio, H. A., Ying, S., Zins, J. L., Patel, K., McKnight, T. D. and Plaxton, W. C. (2012). The secreted purple acid phosphatase isozymes AtPAP12 and AtPAP26 play a pivotal role in extracellular phosphate-scavenging by Arabidopsis thaliana. J Exp Bot 63(18): 6531-6542.