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==Protein Extraction and 2-D PAGE== | ==Protein Extraction and 2-D PAGE== | ||
| − | *A portion (500 mg) of the shoots was removed,homogenized with 1 mL of lysis buffer [2] and centrifuged at 15 000 g for 5 min. The supernatant (50 mL) was subjected to 2-D PAGE [2]. IEF was carried out in a glass capillary tube of 13 cm length and 3 mm diameter. Briefly,the gel solution consisted of 10% NP-40, 30% w/v acrylamide, 9.5 M urea, 10% ammonium persulfate, and an equal mixture of 2% carrier ampholytes (pH 3.5±10and 5±8). The sample overlay buffer consisted of 20 mL of 1/2 lysis buffer. Electrophoresis was carried out at 200 V for 30 min, followed by 400 V for 16 h and 800 V for 1 h.Sodium dodecyl sulfate (SDS)-PAGE in the second dimension was performed with 15% separation gels and 5% stacking gels at a constant current of 35 mA. The | + | *A portion (500 mg) of the shoots was removed,homogenized with 1 mL of lysis buffer [2] and centrifuged at 15 000 g for 5 min. The supernatant (50 mL) was subjected to 2-D PAGE [2]. IEF was carried out in a glass capillary tube of 13 cm length and 3 mm diameter. Briefly,the gel solution consisted of 10% NP-40, 30% w/v acrylamide, 9.5 M urea, 10% ammonium persulfate, and an equal mixture of 2% carrier ampholytes (pH 3.5±10and 5±8). The sample overlay buffer consisted of 20 mL of 1/2 lysis buffer. Electrophoresis was carried out at 200 V for 30 min, followed by 400 V for 16 h and 800 V for 1 h.Sodium dodecyl sulfate (SDS)-PAGE in the second dimension was performed with 15% separation gels and 5% stacking gels at a constant current of 35 mA. The isoelectric point and relative molecular weight of each protein were determined using the Isoelectric Focusing Calibration Kit (pH 3.5±10) from Pharmacia Biotech(Uppsala, Sweden). The localization sites of individual proteins on the gels were evaluated automatically with Image Master 2D Elite software (Pharmacia Biotech). |
| − | isoelectric point and relative molecular weight of each protein were determined using the Isoelectric Focusing Calibration Kit (pH 3.5±10) from Pharmacia Biotech(Uppsala, Sweden). The localization sites of individual proteins on the gels were evaluated automatically with Image Master 2D Elite software (Pharmacia Biotech). | ||
*Following separation by 2-D PAGE, the proteins were electroblotted onto a polyvinylidene difluoride (PVDF)membrane (ProBlott; Applied Biosystems, Foster City,CA, USA) and detected by Ponceau 3R staining [11]. The spots were excised from the PVDF membrane and applied to the upper glass block of the reaction chamber in a gas-phase protein sequencer (477A; Applied Biosystems). Edman degradation was performed according to the standard program supplied by Applied Biosystems.The released phenylthiohydantoin amino acid derivatives were identified by the on-line systems of high performance liquid chromatography (120A; Applied Biosystems). | *Following separation by 2-D PAGE, the proteins were electroblotted onto a polyvinylidene difluoride (PVDF)membrane (ProBlott; Applied Biosystems, Foster City,CA, USA) and detected by Ponceau 3R staining [11]. The spots were excised from the PVDF membrane and applied to the upper glass block of the reaction chamber in a gas-phase protein sequencer (477A; Applied Biosystems). Edman degradation was performed according to the standard program supplied by Applied Biosystems.The released phenylthiohydantoin amino acid derivatives were identified by the on-line systems of high performance liquid chromatography (120A; Applied Biosystems). | ||
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Revision as of 14:20, 17 July 2016
Contents
Project Title
- Separation and characterization of proteins from green and etiolated shoots of rice (Oryza sativa L.):Towards a rice proteome
The Background of This Project
Plant Culture & Treatment
- Green and etiolated shoots of rice (Oryza sativa L. cultivar Nipponbare) were used for this study. Rice seeds were sown on vermiculite and incubated at 25oC under light for 12 h and in the dark for 12 h (for green shoots) or in the dark for 24 h (for etiolated shoots)
Protein Extraction and 2-D PAGE
- A portion (500 mg) of the shoots was removed,homogenized with 1 mL of lysis buffer [2] and centrifuged at 15 000 g for 5 min. The supernatant (50 mL) was subjected to 2-D PAGE [2]. IEF was carried out in a glass capillary tube of 13 cm length and 3 mm diameter. Briefly,the gel solution consisted of 10% NP-40, 30% w/v acrylamide, 9.5 M urea, 10% ammonium persulfate, and an equal mixture of 2% carrier ampholytes (pH 3.5±10and 5±8). The sample overlay buffer consisted of 20 mL of 1/2 lysis buffer. Electrophoresis was carried out at 200 V for 30 min, followed by 400 V for 16 h and 800 V for 1 h.Sodium dodecyl sulfate (SDS)-PAGE in the second dimension was performed with 15% separation gels and 5% stacking gels at a constant current of 35 mA. The isoelectric point and relative molecular weight of each protein were determined using the Isoelectric Focusing Calibration Kit (pH 3.5±10) from Pharmacia Biotech(Uppsala, Sweden). The localization sites of individual proteins on the gels were evaluated automatically with Image Master 2D Elite software (Pharmacia Biotech).
- Following separation by 2-D PAGE, the proteins were electroblotted onto a polyvinylidene difluoride (PVDF)membrane (ProBlott; Applied Biosystems, Foster City,CA, USA) and detected by Ponceau 3R staining [11]. The spots were excised from the PVDF membrane and applied to the upper glass block of the reaction chamber in a gas-phase protein sequencer (477A; Applied Biosystems). Edman degradation was performed according to the standard program supplied by Applied Biosystems.The released phenylthiohydantoin amino acid derivatives were identified by the on-line systems of high performance liquid chromatography (120A; Applied Biosystems).
Research Findings
Labs working on this Project
- Department of Molecular Genetics, National Institute of Agrobiological Resources,Tsukuba, Japan
Corresponding Author
- Dr. Setsuko Komatsu:skomatsu@abr.affrc.go.jp
