Abstract
This protocol describes the isolation of tonoplast vesicles from tomato fruit. The vesicles isolated using this procedure are of sufficiently high purity for downstream proteomic analysis whilst remaining transport competent for functional assays. The methodology was used to study the transport of amino acids during tomato fruit ripening (Snowden et al., 2015) and based on the procedure used by Betty and Smith (Bettey and Smith, 1993). Such vesicles may be useful in further studies into the dynamic transfer of metabolites across the tonoplast for storage and metabolism during tomato fruit development.
Materials and Reagents
Equipment
Procedure
The core procedure detailed here was designed to isolate high purity tonoplast vesicles for proteomic analysis. Modifications to the step gradient are included that allow higher yield preparations suitable for transport assays. As with all organelle isolation procedures the balance between yield and purity will depend on the downstream application. For example proteomic approaches are facilitated by a higher purity of membranes whilst transport assays require a higher yield of membrane. The scale of this protocol can be readily adapted for between 20 g to 80 g of starting material, maintaining the same proportion of plant biomass to buffer. This procedure is carried out at 4 °C, in a cold room, using pre-chilled equipment and freshly prepared solutions incubated on ice for 4 h before starting.
Representative data
Figure 3. Assessment of vesicle purity and integrity. Membrane fraction purities from full A and simplified B gradients were assessed by the inhibition of ATPase activities by 500 μM sodium orthovanadate (inhibits plasma membrane ATPase), 500 μM sodium azide (inhibits mitochondrial ATPase), or 50 mM KNO3 (inhibits tonoplast ATPase). C. Tonoplast vesicle integrity from the 6 24% membrane fraction of the gradient in panel B was assessed by the increase in ATPase activity in the presence of the detergent Brij® 58 (final concentration 0.015 mg ml-1).
Notes
Recipes
Acknowledgments
The research leading to the development of this protocol was funded by the Biotechnology and Biological Sciences Research Council, UK (grant BB/H00338X/1) and Syngenta.
References
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