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A Transient Expression Assay Using Arabidopsis Mesophyll Protoplasts
Published: May 20, 2011 DOI: 10.21769/BioProtoc.70 Views: 21261
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Abstract
This method can be used to free and separate the mesophyll cells from Arabidopsis leaves. The protoplasts that are generated in this way can be used for transient expression for protein activity and subcellular localization assays.
Keywords: Protoplast
Transient expression
Arabidopsis
Transfection
Materials and Reagents
- Arabidopsis (Ecotype: Columbia)
- Fetal bovine serum (FBS) (Sigma-Aldrich, catalog number: F6178 )
- PEG4000 (Fluka, catalog number: 81240 )
- Mannitol
- NaCl
- KCl
- CaCl2
- MgCl2
- 0.5 M MES (pH 5.7)
- β-mercaptoethanol
- Cellulase R10 purchased from http://www.yakult.co.jp/ypi/english/frame03.html
- Macerozyme R10 purchased from http://www.yakult.co.jp/ypi/english/frame03.html
- Bright-Line/Dark-Line Counting Chambers (catalog numbers: 3100 , 3110 , 3200 , 3500 , 1490 , 1492 , 1475 and 1483 )
- Enzyme solution (see Recipes)
- PEG solution (see Recipes)
- W5 solution (see Recipes)
- MMg solution (see Recipes)
- Washing and incubation solution (see Recipes)
Equipment
- IEC clinical centrifuge
- Chamber Counter (instruction attached)
- Petri plate
- Vacuum desiccator
- Zeiss LSM510
- Aluminum foil
- Nylon filters (35-75 µm) (Carolina Biological Supplies, catalog number: 65-2222N )
Procedure
Category
Molecular Biology > Protein > Expression
Plant Science > Plant molecular biology > Protein
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