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http://www.mitosciences.com PRODUCTS FOR RESEARCH INTO THE FUNCTION AND DYSFUNCTION OF MITOCHONDRIAL COMPLEX III MS301 COMPLEX III IMMUNOCAPTURE KIT Isolates Complex III from human, mouse, rat and bovine tissues and cell lines immunoprecipitates obtained. The subunits of the complex are clearly resolved. RESEARCH USES The Complex III immunocapture kit allows isolation of the ubiquinol-cytochrome c reductase complex (E.C. 1.10.2.2) from small amounts of tissue. This facilitates subsequent analysis of assembly state, activity and the extent of post translational modifications including oxidative damage that occur with aging. Uses for the Complex III immunocapture kit include research on genetic mitochondrial disease (1), where complex III may have an additional role in the stabilization of complex I (2). DESCRIPTION The key component of the Complex III immunocapture kit is a monoclonal antibody able to selectively immunocapture the enzyme complex. The mAb is already covalently cross linked to Protein G-Agarose for convenience of use. This material is provided in batches of 25, 50 and 75 l beads which have been charged with approximately 250, 500 and 750 g of antibody respectively. When used as described in the protocol in Figure 1, 10 l of beads are able to immunocapture approximately 10 g of Complex III from heart mitochondria. Also provided are 2 mg of bovine heart mitochondria as a positive control to be used prior to, or during, isolation of Complex III from experimental samples. As an alternative, researchers can purchase the individual components i.e. 100 g of mAb and 2 mg BHM (kit MS301c). SUGGESTED PROTOCOL FOR IMMUNOCAPTURING COMPLEX III The amount of Complex III that is captured in any experiment depends on both the amount of capture antibody and the amount of cell extract or isolated mitochondria used. Calculation of the amount of beads to be used in any experiment must also take account of the source of the material from which Complex III is to be isolated because mitochondria from different tissues have different concentrations of the enzyme complex. For example the levels of Complex III in mitochondria from cell culture material are around 10 fold less that in heart mitochondria. Figure 1 shows a schematic of a generic protocol developed for isolating Complex III from heart tissue. 1mg mitochondria in 0.18 ml Buffer A, 1 g/ml pepstatin, 1g/ml leupeptin, 1mM PMSF Add 20 l 10% LM incubate on ice 30 mins Centrifuge 72000g 4oC 10 mins. Incubate supernatant with 10 l capture resin MS301 3 hours at room temperature OR overnight 4oC Beads collected by gentle centrifugation (10 sec 500g) and washed for 5 mins in PBS, 0.05 % LM, repeat twice Complex III eluted from the resin by addition of 50 l 0.1 M glycine, pH 2.5, 0.05 % LM or 50 l 1 % SDS Protein concentration determined. (e.g. by micro-BCA method (Pierce)) Buffer A : 50 mM Tris.HCl pH 7.5 Leupeptin : 1 mg/ml (water) Pepstatin : 1 mg/ml (ethanol) PMSF : 0.3 M (ethanol) LM : n-dodecyl-b-D-maltoside Figure 1. Example of Complex III isolation procedure from heart mitochondria Figure 2 shows the subunit structure of Complex III immunocaptured from beef heart and human heart mitochondria by Coomassie blue stained SDS-PAGE of MitoScience LLC, 1850 Millrace Drive, Suite 3A, Eugene, OR 97403, +1 (800) 910-6486, (541) 284-1800, Fax (541) 284-1801 Page 1 of 4 http://www.mitosciences.com PRODUCTS FOR RESEARCH INTO THE FUNCTION AND DYSFUNCTION OF MITOCHONDRIAL COMPLEX IV MATERIALS AND STORAGE Kit MS301 contains the anti-Complex III immunocapture mAb covalently linked to protein G-agarose beads in 25, 50 or 75 l amounts. Beads have 8-10 g mAb bound per l bead volume. All volumes of bead resin are suspended in 400 l of PBS buffer (1.4 mM KH2PO4, 8 mM Na2HPO4, 140 mM NaCl, 2.7 mM KCl, pH 7.3) with 0.02 % sodium azide. Also included are 2 mg of purified bovine heart mitochondria resuspended in 400 l of heart mitochondria resuspension buffer (10 mM Tris.HCl, pH 7.8, 0.2 M sucrose, 0.2 mM EDTA, 1 mM PMSF). The antibody is shipped on ice. Upon receipt store the mAb at 4oC. The mitochondrial preparation should be aliquoted and stored at -20oC until use. 1000 g BHM 500 g 500 g BHM HHM 188 98 ADDITIONAL PRODUCTS THAT COMPLEMENT THE COMPLEX III IMMUNOCAPTURE KIT MS302 COMPLEX III SUBUNIT WESTERN BLOTTING KIT Confirms Complex III enzyme purity RESEARCH USES The Complex III Western blotting kit contains three different mAbs reacting specifically with the core protein 1, the Rieske protein, and a 10 kDa Complex III subunit of complex III respectively, along with a mAb against the subunit of Complex V (ATP synthase F1) included as a control. This mAb mixture can be used to probe the isolated complex III for integrity and purity as shown in Figure 1. Alternatively the cocktail can be used to conveniently detect the presence of 3 key Complex III proteins relative to a control (Complex V ) in patient samples. Also included in this kit are SDSdissociated human heart mitochondria to use as a control for the identification of Complex III subunits on the gel system chosen for analysis. 62 Acyl CoA deHase Core 1 Core 2 49 38 ANT Porin 3 4 c1 5 Rieske 28 17 14 6 7 8 9, 10 6 Figure 2. Immunocapture of Complex III from human and bovine mitochondria. Using 10 l of capture resin MS301, the yield of Complex III from 1000 g of bovine heart mitochondria (BHM) is approximately 10 g. However a yield detectable by Coomassie staining can be obtained from as little as 250 g of total mitochondria. This yield is enough for most mass spectrometry applications Figure 3. Complex III subunit identification. MAb cocktail M302 can be used to verify the presence of the core protein 1, Rieske protein and a 10 kDa subunit in immunocaptured Complex III while using the ATP synthase F1subunit mAb as a control. The subunit is an abundant mitochondrial protein. MitoScience LLC, 1850 Millrace Drive, Suite 3A, Eugene, OR 97403, +1 (800) 910-6486, (541) 284-1800, Fax (541) 284-1801 Page 2 of 4 http://www.mitosciences.com PRODUCTS FOR RESEARCH INTO THE FUNCTION AND DYSFUNCTION OF MITOCHONDRIAL COMPLEX IV MATERIALS AND STORAGE Kit MS302 contains a cocktail of antibodies totaling 200 g (MS302-200) or 400 g (MS302-400). The mixture is comprised of 4 mAbs reactive to Complex III Core protein 1 (50 kDa), the Rieske protein (25 kDa) and a 10 kDa subunit of complex III and Complex V in PBS buffer (1.4 mM KH2PO4, 8 mM Na2HPO4, 140 mM NaCl, 2.7 mM KCl, pH 7.3), supplemented with 0.02 % sodium azide. The mAb cocktail is shipped on ice; upon receipt store at 4oC. Also supplied are human heart mitochondria solubilized in SDSPAGE loading buffer (2 % SDS, 60 mM Tris.HCl, pH 6.8, 5 % glycerol, 0.02 mg/ml bromophenol blue). This control mitochondrial sample should be stored at 4oC or for longer periods of time at -20oC. ANTI-COMPLEX III SUBUNIT ANTIBODIES FOR VARIED APPLICATIONS Monoclonal antibodies are available to four Complex III subunits for varied applications such as Western blotting Immunocytochemistry, ELISA of human samples (see Figure 4). MS303 anti-Complex III Core Protein 1 mAb for Western blotting MS304 anti-Complex III Core Protein 2 mAb for Western blotting and Immunocytochemistry (see Figure 5). MS305 anti-Complex III FeS (Rieske protein) mAb for Western blotting MS306 anti-Complex III-10 kD mAb for Western blotting. Figure 5. Immunocytochemical labeling of human MRC5 fibroblast mitochondria with MS304 an anti Complex III core 2 antibody. In this example the anti-Core 2 mouse monoclonal antibody is visualized by the addition of an Alexa 488 Goat anti mouse antibody (Molecular Probes Inc). MATERIALS AND STORAGE These products contain 100 g of mAb (MS303-306) dissolved in 100 l of PBS, 0.02% azide. The mAb is shipped on ice; upon receipt store at 4oC. Also supplied as a Western blot control sample are human heart mitochondria solubilized in SDS-PAGE loading buffer (2 % SDS, 60 mM Tris.HCl, pH 6.8, 5 % glycerol, 0.02 mg/ml bromophenol blue). This control mitochondrial sample should be stored at 4oC or for longer periods of time at -20oC. (MS601) WESTERN BLOTTING DETECTION KIT FOR OXPHOS COMPLEXES A mAb cocktail optimized for the detection of a subunit from each of the 5 OXPHOS complexes RESEARCH USES Figure 4. Western blotting using complex III monoclonal antibodies. This kit is an already mixed solution containing 5 mAbs, one each against a 20 kDa subunit of Complex I, 30 kDa nonheme iron subunit of succinate dehydrogenase (Complex II), core protein 2 of Complex III, subunit II of cytochrome c oxidase (Complex IV) and the subunit of the ATP synthase (Complex V). These polypeptides are well separated in SDS- MitoScience LLC, 1850 Millrace Drive, Suite 3A, Eugene, OR 97403, +1 (800) 910-6486, (541) 284-1800, Fax (541) 284-1801 Page 3 of 4 http://www.mitosciences.com PRODUCTS FOR RESEARCH INTO THE FUNCTION AND DYSFUNCTION OF MITOCHONDRIAL COMPLEX IV Cocktail V F1 III II IV core 2 Ip 30kDa COXII I 20kDa PAGE as shown in Figure 6. The OXPHOS mAb cocktail provides an important analysis of the purity of OXPHOS complexes after immunocapture. However this cocktail could also be used to detect a specific reduction in the levels of an individual OXPHOS complex with respect to the others. Also included are SDS-dissociated human heart mitochondria as an SDS-PAGE control. MATERIALS AND STORAGE Figure 6. Simultaneous blotting of a representative subunit from all 5 OXPHOS complexes. In this figure each lane contains 10 g of MRC5 fibroblast mitochondria. mAb cocktail M601 is shown in the first lane and for demonstrative purposes the individual mAbs making up this cocktail are shown alongside, these are a 20 kDa subunit (Complex I), 30 kDa Ip subunit (Complex II), core 2 (Complex III), COX II (Complex IV) and F1 (ATP synthase) respectively. Kit MS601 is available in 300 g (MS601-300) or 600 g (MS601-600) amounts in PBS (0.02% azide) of each of 5 mAbs against I-20 kDa, II-30 kDa, III-core2, IV-COXII and ATP synthase F1. The mAb cocktail is shipped on ice; upon receipt store at 4oC. Also supplied are human heart mitochondria solubilized in SDS-PAGE loading buffer (2 % SDS, 60 mM Tris.HCl, pH 6.8, 5 % glycerol, 0.02 mg/ml bromophenol blue). This control mitochondrial sample should be stored at 4oC or for longer periods of time at -20oC. 1. 2. J Clin Invest (2003) 111, 303-312 Mol Cell (2004) 13, 805-815 Price List MS301-250 Kit to immunocapture ~25 g of Complex III from heart mitochondria plus 2 mg of BHM control. . . . . . . . . . . . . . . . . . . . . . . . $295 MS301c contains 100 g of Complex III immunocapture antibody plus 2 mg of BHM control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. $270 MS301-500 Kit to immunocapture ~50 g of Complex III from heart mitochondria plus 2 mg of BHM control. . . . . . . . . . . . . . . . . . . . . . . . .$550 MS301-750 Kit to immunocapture ~75 g of Complex III from heart mitochondria plus 2 mg of BHM control. . . . . . . . . . . . . . . . . . . . . . . . .$795 MS302-200 Complex-III Subunit Western Blotting Kit containing a mixture of mAbs totaling 200 g plus HHM control . . . . . . . . . . . . . . . . $375 MS302-400 Complex-III Subunit Western Blotting Kit containing a mixture of mAbs totaling 400 g plus HHM control . . . . . . . . . . . . . . . . $695 MS303 Western Blotting Detection of Core-1 (100 g mAb) plus HHM control. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . $250 MS304 Western Blotting Detection of Core-2 (100 g mAb) plus HHM control. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . $250 MS305 Western Blotting Detection of Rieske FeS protein (100 g mAb) plus HHM control. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .$250 MS306 Western Blotting Detection of a Complex III 10 kDa subunit (100 g mAb) plus HHM control. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . $250 MS601-300 Total OXPHOS Complexes Western Blotting Detection Kit containing a mixture of mAbs totaling 300 g plus HHM control. . . $375 MS601-600 Total OXPHOS Complexes Western Blotting Detection Kit containing a mixture of mAbs totaling 600 g plus HHM control. . . $695 All MitoSciences products are sold “FOR RESEARCH PURPOSES ONLY” MitoScience LLC, 1850 Millrace Drive, Suite 3A, Eugene, OR 97403, +1 (800) 910-6486, (541) 284-1800, Fax (541) 284-1801 Page 4 of 4