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. Author manuscript; available in PMC: 2012 Aug 9.

Published in final edited form as: Circulation. 2011 Jul 18;124(6):682–694. doi: 10.1161/CIRCULATIONAHA.111.023259

A. Site-directed fluorescence labeling of the RyR2 with MCA. A. MCA fluorescence labeling took place specifically on RyR2 (fluorescence image, left upper panel) of many SR proteins (CBB protein staining, right upper panel), by using DP2246 as the carrier. MCA fluorescence was barely detected when either an excess concentration of DP2246 (10 mmol/L) (‘cold chase’) or Ab2141 (antibody against the K201-binding domain) was added during the labeling, indicating that DP2246-mediated fluorescent labeling was site-specific. Three exogenous proteins with Mr (in kDa) 250, 150 and 100 were used as Mr markers. CBB: Coomassie Brilliant Blue.

B. Identification of the partner domain of DP2246 in the tryptic fragments of the fluorescence labeled RyR2. Using DP2246 as the site-directed carrier, MCA fluorescence labeling took place within RyR2 in SR vesicles. After tryptic digestion of the fluorescently labeled RyR2, the 60 kD fragment was detected as the shortest MCA-labeled segment of the RyR2 by Ab2141. Ab2141; antibody against K201-binding domain (epitope ²¹³⁴MGKEEEKLMIRGLGDI²¹⁴⁹). Ab12: antibody against N-terminal region (epitope⁶ EGEDEIQFLRTDDE¹⁹); Ab2808: antibody against phosphorylation site (epitope ²⁸⁰¹YNRTRRISQT²⁸¹⁰); Ab4963: antibody against C-terminal region (epitope ⁴⁹⁵⁹RKQYEDQLN⁴⁹⁶⁷). C. Identification of the partner domain of DP2246 by site-specific MCA labeling of recombinant RyR2 fragments. Of several recombinant RyR2 fragments (1–610, 741–1260, 1245–1768, 1741–2270, 2234–2750), only the fragment1741–2270 was specifically MCA-labeled.

A. Site-directed fluorescence labeling of the RyR2 with MCA. A. MCA fluorescence labeling took place specifically on RyR2 (fluorescence image, left upper panel) of many SR proteins (CBB protein staining, right upper panel), by using DP2246 as the carrier. MCA fluorescence was barely detected when either an excess concentration of DP2246 (10 mmol/L) (‘cold chase’) or Ab2141 (antibody against the K201-binding domain) was added during the labeling, indicating that DP2246-mediated fluorescent labeling was site-specific. Three exogenous proteins with Mr (in kDa) 250, 150 and 100 were used as Mr markers. CBB: Coomassie Brilliant Blue.

B. Identification of the partner domain of DP2246 in the tryptic fragments of the fluorescence labeled RyR2. Using DP2246 as the site-directed carrier, MCA fluorescence labeling took place within RyR2 in SR vesicles. After tryptic digestion of the fluorescently labeled RyR2, the 60 kD fragment was detected as the shortest MCA-labeled segment of the RyR2 by Ab2141. Ab2141; antibody against K201-binding domain (epitope ²¹³⁴MGKEEEKLMIRGLGDI²¹⁴⁹). Ab12: antibody against N-terminal region (epitope⁶ EGEDEIQFLRTDDE¹⁹); Ab2808: antibody against phosphorylation site (epitope ²⁸⁰¹YNRTRRISQT²⁸¹⁰); Ab4963: antibody against C-terminal region (epitope ⁴⁹⁵⁹RKQYEDQLN⁴⁹⁶⁷). C. Identification of the partner domain of DP2246 by site-specific MCA labeling of recombinant RyR2 fragments. Of several recombinant RyR2 fragments (1–610, 741–1260, 1245–1768, 1741–2270, 2234–2750), only the fragment1741–2270 was specifically MCA-labeled.