title: A rapid and reliable flow cytometric method for determining Hsp70 levels in tobacco protoplasts.
abstract: Current methods to determine heat shock protein (Hsp) synthesis or accumulation in plant cells, such as Western blotting and biometabolic labelling are either indirectly quantitative, labour-intensive or biohazardous. An optimal flow cytometric protocol was developed to measure the intracellular Hsp70/Hsc70 levels in tobacco protoplasts. After heat treatments, protoplasts were fixed in 2% paraformaldehyde-phosphate-buffered saline and dehydrated overnight in methyl cellusolve, followed by permeabilization with Triton X-100 (0.1% in Protoplast Wash Fluid). Immunolabelling of Hsp70/Hsc70 was done for 1 hour with a mouse monoclonal antibody and detected by R-Phycoerythrin-conjugated goat anti-mouse IgG using flow cytometry. Flow cytometry detected a significant 1.2-fold increase in Hsp70/Hsc70 accumulation (P < 0.001) in protoplasts, while Western blotting, quantified by image analysis, showed induction under similar conditions but at lower significance (P < 0.05). The coefficients of variance for flow cytometry and Western blotting were 30.7 and 49.8 respectively. Optimum temperature of heat-induced Hsp70/Hsc70 accumulation in tobacco protoplasts occurred at 40 degrees C. Flow cytometry is proposed as a quantitative, more reproducible and rapid alternative to Western blotting for the detection of Hsp70 accumulation in plant cells.
authors.affiliation: Department of Chemistry and Biochemistry, RAU University, Auckland Park, South Africa. mjc@na.rau.ac.za
authors.forename: Marianne J
authors.initials: MJ
authors.lastname: Cronjé
authors.forename: Marisha
authors.initials: M
authors.lastname: Snyman
authors.forename: Liza
authors.initials: L
authors.lastname: Bornman
authors.forename: Iona E
authors.initials: IE
authors.lastname: Weir
mesh_terms: D015153:Blotting, Western; D005434:Flow Cytometry / Q000379:methods; D018506:Gene Expression Regulation, Plant / Q000502:physiology*; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism*; D011523:Protoplasts / Q000378:metabolism*; D013696:Temperature; D014026:Nicotiana / Q000378:metabolism*
chemical_list: D018840:HSP70 Heat-Shock Proteins