shown in Fig. 2a, radioactivities released from the con- trol vesicles were very low and essentially constant within the experimental period indicating that the leakage of 45 Ca 2+ incorporated into the sealed vesicles is, if any, negligible even in the presence of Brij 58. In contrast, appreciable amounts of 45 Ca 2+ were dis- charged from the vesicles when physiological concen- tration of cyclic AMP (3) or valinomycin was added to the suspension of the vesicles (Fig. 2a). Approximately 31 and 33% of 45 Ca 2+ in the sealed vesicles were re- leased by the treatment with cyclic AMP and valino- mycin, respectively, suggesting that the activities of the cyclic nucleotide-dependent and voltage-dependent Ca 2+ channels were not lost by the treatment with the detergent. In order to compare the activities of Ca 2+ channels in the absence and presence of Brij 58, 45 Ca 2+ - loaded vesicles were similarly prepared and treated with cyclic AMP and valinomycin in the absence of the detergent (Fig. 2b). Although a fluctuation of the ra- dioactivities was observed in the detergent-free vesi- cles, 39 and 34% 45 Ca 2+ were released by the treatment with cyclic AMP and valinomycin, respectively. Even if considering the unstable background radioactivities, these figures appeared to be similar to those obtained in the Brij 58-treated vesicles (Fig. 2a). Since 45 Ca 2+ is loaded into and discharged from solely inside-out-ori- ented vesicles under the detergent-free condition, it is reasonable to conclude that activities of plasma mem- brane-located Ca 2+ channels stimulated by cyclic AMP or K + flux are not reduced even in the presence of 0.2% Brij 58. In conclusion, determination of 45 Ca 2+ discharged from the ion-loaded sealed vesicles of plant plasma membrane reoriented with Brij 58 is a convenient method to assess Ca 2+ influx triggered by the changes in inter- and intracellular conditions of higher plants. Environments inside and outside of intact plant cells can be readily mimicked appropriately, and the signal/ noise ratio (the ratio of discharged 45 Ca 2+ to “back- ground” radioactivities) has been greatly increased by the reorientation of the vesicles without inactivation of the channel proteins. Acknowledgment. This work was supported in part by a Grant- in-Aid from the Ministry of Education, Science and Culture, Japan. REFERENCES 1. Mulligan, R. M., Chory, J., and Ecker, J. R. (1997) Signaling in plants. Proc. Natl. Acad. Sci. USA 94, 2793–2795. 2. Schroeder, J. I., and Thuleau, P. (1991) Ca 2+ channels in higher plant cells. Plant Cell 3, 555–559. 3. Kurosaki, F., and Nishi, A. (1993) Stimulation of calcium influx and calcium cascade by cyclic AMP in cultured carrot cells. Arch. Biochem. Biophys. 302, 144 –151. 4. Kurosaki, F. (1997) Role of inward K + -channel located at carrot plasma membrane in signal cross-talking of cyclic AMP with Ca 2+ -cascade. FEBS Lett. 408, 115–119. 5. Graef, P., and Weiler, E. W. (1989) ATP-driven Ca 2+ -transport in sealed plasma membrane vesicles prepared by aqueous two-phase partitioning from leaves of Commelia communis. Physiol. Plant. 75, 469 – 478. 6. Johansson, F., Olbe, M., Sommarin, M., and Larsson, C. (1995) Brij 58, a polyoxyethylene ether, creates membrane vesicles of uniform sidedness: A new tool to obtain inside-out (cytoplasmic side-out) plasma membrane vesicles. Plant J. 7, 165–173. 7. Murashige, T., and Skoog, F. (1962) A revised medium for rapid growth and bioassay with tobacco tissue cultures. Physiol. Plant. 72, 248 –254. 8. Clough, S. J., Fengler, K. A., Yu, I., Lippok, B., Smith, R. K., and Bent, A. F. (2000) The Arabidopsis dnd1 “defense, no death” gene encodes a mutated cyclic nucleotide-gated ion channel. Proc. Natl. Acad. Sci. USA 97, 9323– 9328. Calsequestrin Blot Overlay of Two-Dimensional Electrophoretically Separated Microsomal Proteins from Skeletal Muscle Louise Glover, Gabriele Froemming, and Kay Ohlendieck 1 Department of Pharmacology, Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Belfield, Dublin 4, Ireland Received June 25, 2001; published online November 10, 2001 Calsequestrin represents a medium-affinity, high- capacity Ca 2+ -binding protein of the sarcoplasmic re- ticulum (1, 2) and plays a central role in the regulation of the excitation– contraction–relaxation cycle in skel- etal muscle (3, 4). Although the physiological and bio- chemical characterization of this terminal cisternae protein (5–7), as well as electron microscopical studies (8, 9), strongly suggest that calsequestrin forms large protein aggregates, no direct biochemical evidence ex- ists demonstrating protein–protein interactions within triadic Ca 2+ -binding units. In determining protein linkage biochemically, one-dimensional (1D) 2 gel elec- trophoretic separation and blotting methodology em- ploying purified proteins have been established as effective tools. However, interactions between immobi- lized calsequestrin and purified calsequestrin failed in a previous blot overlay attempt (10). Only low molecu- lar mass calsequestrin-binding proteins such as junc- tin were identified (10). Thus, to demonstrate direct coupling between calsequestrin monomers of apparent 63 kDa, we advanced the overlay blotting technique in the following manner: (a) instead of 1D blots, nitrocel- 1 To whom correspondence and reprint requests should be ad- dressed. E-mail: kay.ohlendieck@ucd.ie. 2 Abbreviations used: 1D, one-dimensional; 2D, two-dimensional; CSQ, calsequestrin. 268 NOTES & TIPS Analytical Biochemistry 299, 268 –271 (2001) doi:10.1006/abio.2001.5424 0003-2697/01 $35.00 © 2001 Elsevier Science All rights reserved.