A rapid chemical method for lysing Arabidopsis cells for protein analysis
© Tsugama et al; licensee BioMed Central Ltd. 2011
Received: 17 June 2011
Accepted: 15 July 2011
Published: 15 July 2011
Protein extraction is a frequent procedure in biological research. For preparation of plant cell extracts, plant materials usually have to be ground and homogenized to physically break the robust cell wall, but this step is laborious and time-consuming when a large number of samples are handled at once.
We developed a chemical method for lysing Arabidopsis cells without grinding. In this method, plants are boiled for just 10 minutes in a solution containing a Ca2+ chelator and detergent. Cell extracts prepared by this method were suitable for SDS-PAGE and immunoblot analysis. This method was also applicable to genomic DNA extraction for PCR analysis. Our method was applied to many other plant species, and worked well for some of them.
Our method is rapid and economical, and allows many samples to be prepared simultaneously for protein analysis. Our method is useful not only for Arabidopsis research but also research on certain other species.
KeywordsCell wall pectin Ca2+ Chelate detergent Arabidopsis
Protein extraction is a frequent procedure in biological research. For preparation of plant cell extracts, it is usually required to grind and homogenize plant materials to physically break the robust cell wall. Sample grinding is laborious and time-consuming, especially when a large number of samples are handled at once. In the case of yeast cells, which also have a cell wall, proteins can be efficiently extracted after cells are treated with alkaline (NaOH) and boiled in SDS-containing solution [1–3]. Although the components and structure of the yeast cell walls [4, for a review] are different from those of the plant cell wall [5, for a review], the simplicity of the yeast method tempted us to seek out a chemical cell-lysis method suitable for plant protein extraction.
Here we describe a rapid and simple way of preparing cell extracts from Arabidopsis. We found that in the presence of certain amounts of a Ca2+ chelator and detergent, Arabidopsis cells are quickly lysed just by boiling, without grinding. The method is rapid and economical, and the cell extracts prepared by the method are suitable for SDS-PAGE and immunoblot analysis.
Results and Discussion
Effects of Ca2+-chelating agents on Arabidopsis cell lysis
Chelators used in this study
logK CaL * 1
Rapid preparation of cell extracts
Applicability of the method to different tissues and plants
We have developed a chemical method for lysing unground Arabidopsis cells, which is based on cell wall loosening by a Ca2+ chelator and on membrane solubilization by detergent. Our method is applicable to some other species, and cell extracts prepared by our method is suitable for SDS-PAGE, immunoblot and genomic PCR analysis. Our method is rapid, economical, and thus useful for plant research.
EDTA (EDTA disodium salt dihydrate) was purchased from Bio-Rad (Japan), EGTA from Sigma (USA), NTA (nitrilotriacetic acid), TTHA (triethylenetetramine-N, N, N', N", N"', N"'-hexaacetic acid), DTPA (diethylenetriamine-N, N, N', N", N"-pentaacetic acid), CyDTA (trans-1,2-diaminocyclohexane-N, N, N', N'-tetraacetic acid, monohydrate) and BAPTA (O,O'-bis(2-aminophenyl)ethyleneglycol-N, N, N', N'-tetraacetic acid, tetrapotassium salt, hydrate) from Dojindo (Japan). All these chelating agents except EDTA were prepared as 100 mM stock solutions by diluting them in 0.4 M NaOH. EDTA was prepared as a 0.5 M stock solution of pH 8.0.
SDS, SDSa and Tween 20 were purchased from Wako (Japan), CHAPS from Dojindo, and Brij 35 from Calbiochem. SDS, SDSa and CHAPS were prepared as 10% w/v stock solutions in distilled water, Tween 20 and BriJ 35 as 10% v/v. SDS was used in the concentrations described in the figures. SDSa and CHAPS were used in the final concentrations of 0.1-1% w/v. Tween 20 and Brij 35 were used in the final concentration of 0.1-1% v/v.
Plant materials, growth conditions and plant transformation
Arabidopsis thaliana ecotype Columbia-0 (Col-0) was used throughout the experiments. Seeds of agb1-2 were obtained from the Arabidopsis Biological Resource Center (ABRC, http://www.arabidopsis.org). Surface-sterilized seeds were sown on the 0.5× MS medium (0.8% w/v agar, 0.5× MS salts, 1% w/v sucrose, 0.5 g/l MES, pH 5.8) for immunoblot and genomic PCR analyses, or in rockwool cubes for the other experiments. In either case, plants were grown at 22°C under a 12-h light/12-h dark photoperiod. To generate transgenic plants expressing GFP, the GUS coding sequence in pBI121 was replaced with the GFP coding sequence, and this construct was used for Arabidopsis transformation by the Agrobacterium-mediated floral-dip method . GFP expression in T2 plants was checked by fluorescence microscopy, and GFP-positive plants were used for the immunoblot analysis. Plants other than Arabidopsis were grown in the field and healthy-looking leaves were sampled.
Preparation of Arabidopsis cell extracts
All the compositions of the solutions and the detailed procedures for the cell lysis experiments are described in each figure. All the lysis solutions were made by mixing stock solutions of their components. For example, the lysis solution for SDS-PAGE in Figure 5 (0.1 M EDTA, 0.12 M Tris-HCl, 4% w/v SDS, 10% v/v β-ME, 5% v/v glycerol and 0.005% w/v bromophenol blue) was prepared by mixing 0.5 M EDTA, pH 8.0, 1 M Tris-HCl, pH 6.8, 10% w/v SDS, 100% β-ME, 100% glycerol and bromophenol blue powder to give the final concentrations. Images were processed with Canvas X software (ACD Systems).
SDS-PAGE, immunoblot analysis and genomic PCR analysis
SDS-PAGE and immunoblot analysis were carried out according to standard procedures [6, 15]. After CBB staining, signals strengths were quantified using ImageJ software http://rsb.info.nih.gov/ij/index.html. For GFP detection, 7-day-old seedlings grown on the 0.5× MS medium were used. For detection of MAPK activation, 14-day-old seedlings were incubated at room temperature for 20 min in 20 mM Tris-HCl, pH 6.8 with or without 1 μM flg22 or 40 mM H2O2. Immunoblot analysis was performed using an anti-GFP (MBL, Japan), anti-MPK6 (Sigma) or anti-phospho-p44/42 MAPK (Erk1/2) (Cell Signaling Technology, USA) as a primary antibody, and a horseradish peroxidase-conjugated anti-rabbit IgG (MBL) as the secondary antibody. Signals were detected using SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific, USA) and LAS-1000 plus image analyzer (Fuji Film, Japan). After detecting GFP, total proteins on the membrane were stained with Ponceau S solution (0.2% w/v Ponceau S in 5% acetic acid).
For genomic PCR analysis, genomic DNA was prepared from 7-day-old seedlings by the procedure described in Figure 5. PCR was performed using KOD FX (Toyobo, Japan). For 50 μl PCR mixture, 1-3 μl of the template solution was added. Primer sequences were as follows: AGB1 forward, 5'-AGACGCCTCCAGCTCCTCGA-3'; AGB1 reverse, 5'-GCACTTCCATCTGCTGACAACCCC-3'; T-DNA left border, 5'-CAGGATTTTCGCCTGCTGGGGC-3'. PCR cycle: 98°C 2 min, 40 cycles of (98°C 10 sec, 60°C 30 sec, 72°C 1 min), 72°C 7 min, 4°C until analysis. Images were processed with Canvas X software (ACD Systems).
sodium dodecyl sulfate
Coomassie brilliant blue
green fluorescent protein
mitogen-activated protein kinase
triethylenetetramine-N, N, N', N", N"', N"'-hexaacetic acid
diethylenetriamine-N, N, N', N", N"-pentaacetic acid
- CyDTA (trans-1:
2-diaminocyclohexane-N, N, N', N'-tetraacetic acid
O, O'-bis(2-aminophenyl)ethyleneglycol-N, N, N', N'-tetraacetic acid
Murashige and Skoog
2-(4-Morpholino)ethane sulfonic acid.
This work was supported by a Grant-in-aid for Scientific Research (21380002) to T.T. and (22-2144) to D.T. We appreciate the kind help of Dr. Daisuke Takata in sampling the field-grown plants. We are grateful to ABRC for providing the Arabidopsis mutant seeds.
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