Supporting Information Wiley-VCH 2008 69451 Weinheim, Germany
Electronic Supporting Information A Highly Selective Luminescence Switch-on Probe to Histidine/Histidine-rich proteins and Its Application in Protein Staining Dik-Lung Ma,* Wing-Leung Wong, Wai-Hong Chung, Fung-Yi Chan, Pui-Kin So, Tat-Shing Lai, Yun- Chung Leung and Kwok-Yin Wong* Experimental Section Materials. All chemicals and solvents (AR grade) for syntheses were used as received. Bovine serum albumin (BSA, product no. A8531) was purchased from Sigma Chemical Co. Ltd. and used without further purification. Broad-range molecular weight protein standards (Catalog no. 161-0317) and all chemicals for SDS-PAGE were purchased from Bio-Rad. Iridium(III) trichloride hydrated, 2-(2- methoxyethoxy)ethanol, 2-phenyloyridine were obtained from Aldrich. [Ir(ppy) 2 (OH 2 ) 2 ] (1 CF 3 SO 3 ) and Ir(ppy) 3 (2) were prepared according to reported procedures. 1,2 6 His-tagged PenPC β-lactamase and the non-his-tagged PenPC β-lactamase were expressed and purified according to reported procedure. 3 Physical Measurements. Emission spectra were recorded on a Perkin-Elmer LS50B spectrophotometer. 1 H NMR spectra were recorded on a Bruker DPX-400 NMR spectrometer. Electrospray ionization positive-ion mass spectra were recorded on a Hybrid Quadrupole - time of flight [ ] Dr. Dik-Lung Ma, Dr. Wing-Leung Wong, Wai-Hong Chung, Fung-Yi Chan, Pui-Kin So, Dr. Tat-Shing Lai, Prof. Yun-Chung Leung, Prof. Kwok-Yin Wong Department of Applied Biology and Chemical Technology The Hong Kong Polytechnic University Hung Hom, Kowloon, Hong Kong, China Fax: +(852)2364 9932 E-mail: bcedmond@polyu.edu.hk E-mail: bckywong@inet.polyu.edu.hk [ ] This work was supported by The Hong Kong Polytechnic University, and the Area of Excellence Fund of the University Grants Committee (AoE/P-10/01). DLM and WLW acknowledge the award of postdoctoral fellowships administered by the Research Committee of the Hong Kong Polytechnic University. We are grateful to Prof. Chi-Ming Che for supporting this work and for his invaluable suggestions. 1
mass spectrometer mass spectrometer (Q-TOF 2) equipped with a z-spray electrospray ionization source (Waters, Manchester, U.K.). Spectroscopic measurement. Solutions of the iridium(iii) complex (50 μm) were prepared in PBS buffer. Aliquots of a millimolar stock BSA solution (0 5 mm) or various natural amino acids (0 200 μm) were then added. Emission spectra were recorded in the 400 800 nm range, after equilibration at 20.0 C for 10 min. SDS-PAGE. SDS-PAGE analysis was conducted according to Bio-Rad Mini-PROTEAN 3 Cell Instruction Manual using a 4% stacking gel and 10% resolving gel. Protein detection. After electrophoresis, the gels were stained with (1 CF 3 SO 3 ) at room temperature. The iridium complexes (5.0 mg) were first dissolved in MeOH (100 μl) followed by addition of H 2 O (19.95 ml). Approximately 20 ml of staining solution is used for a typical mini-gel (5 cm 9 cm 1 mm). The gel is placed into the staining solution and the container is covered with aluminum foil to protect the dye from bright light. The gel is gently agitated for 10 minutes at room temperature using an orbital shaker (50:RPM). After staining, the gel is rinsed in DI H 2 O for 10 15 minutes and viewed using a 300 nm UV transilluminator. Detection and imaging of the gels were conducted using Alpha DigiDoc TM FC Imaging System with Alpha DigiDoc TM AD-1200 software. The intensities of the protein bands were recorded using the auto-spot function. Detection of Proteins on Filter Membranes Following Dot-blotting or Western Transfer. The protein of interest is diluted in TBS (20 mm Tris-HCl, ph 7.5, 500 mm NaCl), then applied directly to a PVDF or nitrocellulose filter membrane. The membrane is washed once with TBS, allowed to air dry and then is floated face down in a solution containing 5.0 mg of (1 CF 3 SO 3 ) in 0.5% methanol of DI H 2 O (20 ml), ph 7.0 with vigorous mixing.. Alternatively, the proteins are first separated by gel electrophoresis and transferred to a PVDF or nitrocellulose filter membrane using standard procedures. The blot is allowed to dry completely, and is then stained by placing it face down in staining solution as 2
described above. The blots are rinsed in DI H 2 O for 10 15 minutes and viewed using a 300 nm UV transilluminator. References (1) Schmid, B.; Garces, F. O.; Watts, R. J. Inorg. Chem. 1994, 33, 9. (2) King, K. A.; Spellane, P. J.; Watts, R. J. J. Am. Chem. Soc. 1985, 107, 1431. (3) New England Biolabs Manual, http://www.neb.com/nebecomm/manualfiles/manuale8000.pdf. 3
Figure S1. X-ray structure of [Ir(ppy) 2 (CH 3 CN) 2 ]ClO 4. 4
Figure S2. Crystal packing diagram of of [Ir(ppy) 2 (S) 2 ] (1 ClO 4 ). 5
Figure S3. Emissive western blot analysis of proteins with (1 CF 3 SO 3 ) as a detecting stain. 6