Monoclonal Antibodies Specific for Oxidative Phosphorylation

Transcription

1 Product Information Revised: 11 Aug 2006 Quick Facts Storage upon receipt: All products, except A21296, A C Desiccate Products A21296, A C Introduction Protect from light Oxidative phosphorylation (OxPhos) occurs in mitochondria and, in mammals, is catalyzed by five membrane-bound protein complexes, namely NADH-ubiquinol oxidoreductase (Complex I), succinate-ubiquinol oxidoreductase (Complex II), ubiquinol cytochrome c oxidoreductase (Complex III), cytochrome c oxidase (COX) (Complex IV), and ATP synthase (Complex V) (Figure 1). The complexes are composed of multiple subunits, some of which are encoded in the mitochondria and others in the nucleus. Assembly of each complex involves a coordinated association of prosthetic groups with the polypeptides. This complicated process requires assembly factors specific for each complex. Defects in assembly of one or more of these complexes contribute to several described mitochondrial diseases and possibly Alzheimer s and Parkinson s diseases. 1 6 Molecular Probes offers a range of subunit-specific antibodies for the study of mitochondrial function (Tables 1 3 and Figure 1), including monoclonal antibodies specific for subunits of human COX (Complex IV) and for representative subunits of human Complexes I, II, III, and V. These monoclonal antibodies serve as important tools for investigating mitochondrial biogenesis and OxPhos-related diseases. Cell lines can be screened for subunit expression levels for each of the OxPhos complexes by simple Western blotting. 7,8 These results can be combined with native gel electrophoresis or sucrose gradient centrifugation to gather additional information regarding the assembly state of the OxPhos complexes. 9 Many of our antibodies against OxPhos subunits may also be used for immunocytochemical analysis. Image analysis of an antibody s staining pattern can reveal the relative expression and localization of a subunit. This approach has been particularly useful for studying OxPhos subunit expression in diseased muscle fibers 10 and for screening Complex IV deficient patients. 11 For detection of these monoclonal antibodies, Molecular Probes offers anti mouse IgG secondary antibodies labeled with fluorophores, biotin, enzymes such as horseradish peroxidase or alkaline phosphatase, NANOGOLD or Alexa Fluor Fluoro- Nanogold 1.4 nm gold clusters, or Captivate ferrofluid. For labeling combinations of mitochondrial targets, the antibodies in this group can be complexed with the reagents in our Zenon Mouse IgG Labeling Kits or isotype-specific secondary antibodies. See our website (probes.invitrogen.com) for more information about these products. Figure 1. The mammalian oxidative phosphorylation system, and specific monoclonal antibodies available from Molecular Probes. MP 06401

2 Table 1. Specifications for Molecular Probes monoclonal antibodies specific for components of the oxidative phosphorylation system. Catalog Number of Antibody Oxidative Complex Subunit Clone Isotype Suggested Conc. for Westerns (μg/ml) Species Specificity * Hum Bov Rat Mou Suggested Conc. for ICC (μg/ml) Fixation Method ICC Fixation Antigen Retrieval Method A kda 20C11 IgG 1,k NR NA NA A kda 3F9 IgG 1,k ND + NR NA NA I (NADH-ubiquinol A kda 20E9 IgG oxidoreductase) 1,k ND + 5 F, (M) NA A kda 21C11 IgG 2b,k ND M, (F) U A kda 17G3 IgG 1,k ± 1 10 F NA A11142 II (succinateubiquinol 70 kda 2E3 IgG 1,k M NA A21345 oxidoreductase) 30 kda 21A11 IgG 2a,k M NA A21362 Core I 16D10 IgG 1,k ND M T A11143 III (ubiquinol Core II 13G12 IgG 1,k NR NA NA cytochrome c A21346 oxidoreductase) FeS 5A5 IgG 2b,k ND + NR NA NA A kda 1H9 IgG 2a,k ND + NR NA NA A6403 I 1D6 IgG 2a,k (M), (F) NA A6404 II 12C4 IgG 2a,k 2 + ± ± 5 (M), (F) NA A21347 IV 10G8 IgG 2a,k M NA A21348 IV 20E8 IgG 2a,k ND ND NR NA NA A21363 Va 6E9 IgG 2a,k ± + 5 F T or U A21349 IV (cytochrome c oxidase; COX) Vb 16H12 IgG 2b,k NR NA NA A21365 VIa-L ** 14A3 IgG 1,k 5 + ± + NR NA NA A21366 VIb 3F9 IgG 1,k NR NA NA A6401 VIc 3G5 IgG 2b,k NR NA NA A21367 VIIa-H/L 6D7 IgG 2a NR NA NA A21368 VIIb 2G7 IgG 1,k NR NA NA A21350 α 7H10 IgG 2b,k M NA A21351 β 3D5 IgG 1,k ND + 1 F, (M) NA A21353 V (ATP synthase, d 7F9 IgG 2b,k ND ± 5 F T or U A21354 F 1 ) OSCP 4C11 IgG 1,k ND 5 10 F, (M) NA A21355 Inhibitor protein *** 5E2 IgG 1,k F NA * Species specificity: Hum, human; Bov, bovine; Mou, mouse; (+), strong reactivity; (±), weak reactivity; ( ), no reactivity. ICC, immunocytochemistry. Fixation method: M, methanol; F, formaldehyde; ( ) = secondary fixation method. Antigen retrieval method: T, Tris buffer without urea; U, buffer with urea. ** Antibody recognizes subunit from bovine liver. Antibody recognizes subunit from both heart and liver. Oligomycin sensitivity conferring protein. NR = not recommended, ND = not determined, NA = not applicable. *** Although not considered a member of Complex V, the ATPase inhibitor protein is associated with this complex. A second protein band (~18 kda) has been observed with this antibody in Western blots of Complex V preparations from human heart tissue. It has been suggested by the supplier that the identity of this protein is Complex IV, subunit IV, and that the reactivity could be tissue specific. We recommend caution when using the antibody for ICC and IHC applications since the antibody could interact with this protein. Monoclonal Antibodies Specific for OxPhos Complex IV (COX) COX catalyzes the transfer of electrons from reduced cytochrome c to molecular oxygen, with a concomitant translocation of protons across the mitochondrial inner membrane. 12,13 This inner mitochondrial membrane bound enzyme is composed of subunits that are encoded in both the mitochondria (subunits I, II, and III) and the nucleus (ten others). To facilitate the study of COX structure and mitochondrial biogenesis, Molecular Probes offers subunit-specific mouse anti OxPhos Complex IV monoclonal antibodies (Tables 1 and 2). The binding specificity of our anti OxPhos Complex IV monoclonal antibody preparations allows researchers to investigate the regulation, assembly, and orientation of COX subunits in a variety of organisms The antibodies have also proven valuable for analyzing human mitochondrial myopathies and related disorders as well as mitochondrial DNA depletion due to drug toxicity. 7,10,19,20,21 Alexa Fluor 488 and Alexa Fluor 594 conjugates of anti OxPhos Complex IV subunit I are also available for direct staining of these COX proteins (Table 3).

3 Table 2. Molecular characteristics of components of the oxidative phosphorylation system for which Molecular Probes offers antibodies. Catalog Number Complex Subunit Molecular Weight * Alternative Name Protein Number Gene Number A kda 42.5 α subcomplex, 9 Q16795 NDUFA9 A kda 30.2 Iron-sulfur protein, 3 O75489 NDUFS3 A31857 I 20 kda 20.0 ND6 protein P03923 MTND6 A kda 15.5 β subcomplex, 6 O95139 NDUFB6 A kda 15.1 α subcomplex, 6 P56556 NDUFA6 A kda 72.7 Flavoprotein P31040 SDHA II A kda 31.6 Iron-sulfur protein P21912 SDHB A21362 Core I 51.6 None P31930 UQCRC1 A11143 Core II 48.5 None P22695 UQCRC2 III A21346 FeS 29.6 Rieske iron-sulfur protein P47985 UQCRFS1 A kda 9.6 Subunit VII P13271 UCRQ A6403 I 57 COX I P00395 MTCO1 A6404 II 25.6 COX II P00403 MTC02 A21347 IV 19.6 COX IV P13073 COX4 A21348 IV 19.6 COX IV P13073 COX4 A21363 Va 16.8 COX Va P20674 COX5A A21349 IV Vb 13.7 COX Vb P10606 COX5B A21365 VIa-L ** 9.5 COX VIa P13182 COX6A1 A21366 VIb 10.0 COX VIb P00429 COX6B A6401 VIc 8.8 COX VIc P09669 COX6C A21367 VIIa-H/L 9.4 COX VIIa P14406 COX7A2 A21368 VIIb 9.2 COX VIIb P24311 COX7B A21350 α 59.8 F 1 complex, α subunit P25705 ATP5A1 A21351 β 56.6 F 1 complex, β subunit P06576 ATP5B A21353 V d 18.4 None O75947 ATP5H A21354 OSCP 23.2 F 1 complex, O subunit P48047 ATP5O A21355 Inhibitor protein (IP) 12.3 IF 1 P01096 ATPI * Molecular weight, in kilodaltons, based upon the human protein sequence unless otherwise noted; many of these proteins show aberrant molecular weights when analyzed by SDS gel electrophoresis. Protein reference numbers are from SWISS-PROT, an annotated protein sequence database, and refer to the human protein sequence, unless otherwise noted. Gene reference numbers are from GenBank. Protein molecular weight and protein number are for the bovine sequence. ** Antibody recognizes subunit from bovine liver. Antibody recognizes subunit from both heart and liver. Oligomycin sensitivity conferring protein. Monoclonal Antibodies Specific for Complexes I, II, III, and V Molecular Probes offers monoclonal antibodies specific for individual subunits of Complexes I, II, III, and V (Tables 1 and 2). When these monoclonal antibodies are used in combination with the antibodies to Complex IV, the relative levels of all OxPhos complexes in normal and diseased tissues can be evaluated. The Table 3. Alexa Fluor dye conjugates of anti-oxphos Complex IV, subunit I antibody. * Catalog Number Fluorophore Abs Em A21296 Alexa Fluor A21297 Alexa Fluor * For best results in immunocytochemistry, an antigen retrieval procedure is required. Approximate absorption (Abs) and fluorescence emission (Em) maxima, in nm, for conjugates. anti OxPhos Complex V subunit α (A21350) and a polyclonal antibody comparable to anti OxPhos Complex V subunit β (A21351) have also been shown to mimic angiostatin, a potent inhibitor of angiogenesis. 22 Related Products: Mitochondrial Porin and Mitochondrial Protein Extracts Mitochondrial porin is an abundant transmembrane protein that forms regulated channels (referred to as voltage-dependent anionic channels, or VDACs) in the outer mitochondrial membrane. 23 It forms a small pore (~3 nm diameter) in the membrane, allowing molecules smaller than ~10 kda to pass. 24 Due to its abundance, porin is often used as a reference standard in Western blots when assaying for other mitochondrial proteins 9,25 and is an effective marker of mitochondrial mass for immunocytochemistry. 21,26 A monoclonal antibody against human porin is available (A31855).

4 For researchers seeking a source of mitochondrial protein standards, Molecular Probes offers human heart mitochondrial proteins for SDS-polyacrylamide gel electrophoresis (M22430), a complete mitochondrial lysate that has tested negative for hepatitis B and C as well as HIV-1 and -2 in serology tests. This mitochondrial protein extract is useful for evaluating new mitochondrial protein preparations in 1-D or 2-D gels and for testing mitochondrial antibodies. Materials Contents Molecular Probes unlabeled monoclonal antibodies specific for subunits of human OxPhos complexes are supplied in unit sizes of 50 μg and 100 μg. To prepare stock solutions of the lyophilized antibodies, reconstitute them in μl of 1X phosphatebuffered saline (PBS), ph 7.4, containing 1% bovine serum albumin (BSA). The Alexa Fluor dye labeled anti OxPhos IV subunit I antibodies are supplied as 1 mg/ml solutions in PBS, 0.1% BSA, 5 mm sodium azide, in unit sizes of 100 μl. The degree of labeling for each conjugate is typically 2 8 molecules per IgG molecule. At the time of preparation, the products are certified to be free of unconjugated dyes. Storage Upon receipt, store the lyophilized antibodies desiccated at 20 C. When properly stored, these products are stable for at least one year. Store antibody solutions for up to six months at 2 6 C with the addition of 2 mm sodium azide. For longer storage, divide the solutions into single-use aliquots and freeze them at 20 C. AVOID REPEATED FREEZING AND THAWING. Upon receipt, store the Alexa Fluor dye labeled anti OxPhos Complex IV subunit I antibodies at 2 6 C. When properly stored, these products are stable for at least six months. For longer storage, divide the solutions into single-use aliquots and freeze them at 20 C. AVOID REPEATED FREEZING AND THAWING, AND PROTECT FROM LIGHT. Applications The OxPhos antibodies bind monospecifically to the corresponding proteins of the human oxidative phosphorylation system (Table 1 and Figure 1). These antibodies are versatile and can be used in a variety of assay formats including solid-phase binding of native proteins, Western blots of denatured proteins, and immunocytochemistry of fixed cells. Because staining protocols vary with application, the appropriate dilution of an antibody should be determined empirically (see Table 1). For the fluorophore-labeled antibodies, a final concentration of 1 10 μg/ml should be satisfactory for most immunocytochemical applications. 27 It is a good practice to centrifuge antibodies briefly in a microcentrifuge to eliminate any protein aggregates that may have formed during storage and thus reduce nonspecific staining. Specifications The OxPhos monoclonal antibodies were prepared against the appropriate OxPhos complexes isolated from bovine heart, bovine liver, and human heart. The following stringent selection criteria were applied during the development of these antibodies: 1) ability of the antibody to detect native protein by particle-concentration fluorescence immunoassay (PCFIA) and enzyme-linked immunosorbent assay (ELISA), 2) monospecificity of the antibody for denatured protein in Western blots of wholecell extracts and isolated mitochondria, and 3) specific localization of the antibody to mitochondria in fixed cultured human cells. The purity and yield of each preparation was assessed with SDS-polyacrylamide gel electrophoresis and quantitative immunoassay specific for mouse IgG of the appropriate isotype. The subunit specificity and immunoglobulin isotype are shown in Table 1. Note: Most of the proteins involved in the oxidative phosphorylation system are integral membrane proteins, which contain a large number of hydrophobic amino acids that are likely to cause the proteins to run aberrantly with respect to molecular weight in SDS-polyacrylamide gel electrophoresis. Antigen Retrieval In immunocytochemistry applications, methanol-fixed or formaldehyde-fixed and permeabilized target cells may require antigen retrieval methods before the target cells are blocked and labeled with antibodies (see Table 1). 1. Perform antigen retrieval by submersing the slide in preheated (95 C) buffer (10 mm Tris, ph 9.5, 5% urea, or 50 mm Tris, ph 7.5, without urea) (see Table 1), in a preheated staining jar and then placing this jar in a boiling-water bath for 20 minutes. Add boiling chips to the boiling-water bath to prevent spattering. 2. Cool the slide to room temperature, then wash the specimen three times for 5 minutes each in PBS. 3. Block the specimen in a blocking solution such as BlockAid blocking solution (B10710) and/or Image-iT FX signal enhancer (I36933) before proceeding with antibody labeling. For fluorescence imaging we also recommend ProLong Gold antifade reagent (P36930, P36934) or ProLong Gold antifade reagent with DAPI (P36931, P36935). 4

5 References 1. Biochim Biophys Acta 1366, 199 (1998); 2. Biochim Biophys Acta 1366, 211 (1998); 3. Curr Opin Cardiol 13, 190 (1998); 4. Ann Neurol 44, S99 (1998); 5. J Neural Transm 105, 855 (1998); 6. Seminars in Liver Disease 18, 237 (1998); 7. Biochim Biophys Acta 1362, 145 (1997); 8. J Biol Chem 276, 8892 (2001); 9. J Biol Chem 276, (2001); 10. Biochim Biophys Acta 1315, 199 (1996); 11. Brain 123, 591 (2000); 12. Science 283, 1488 (1999); 13. Annu Rev Biochem 59, 569 (1990); 14. Biochemistry 30, 3674 (1991); 15. Biochim Biophys Acta 1225, 95 (1993); 16. Methods Enzymol 260, 117 (1995); 17. J Biol Chem 268, (1993); 18. J Biol Chem 266, 7688 (1991); 19. Pediatr Res 28, 529 (1990); 20. Hum Mol Genet 6, 935 (1997); 21. J Hist Cyto 52 in press (2004); 22. Proc Natl Acad Sci USA 98, 6656 (2001); 23. Biochim Biophys Acta 894, 109 (1987); 24. J Biol Chem 273, (1998); 25. Biochim Biophys Acta 1455, 35 (1999); 26. Mol Biol Cell 9, 917 (1998); 27. Short Protocols in Molecular Biology, 2nd Edition, F.M. Ausubel et al., Eds., John Wiley and Sons (1992) pp Product List Current prices may be obtained from our Web site or from our Customer Service Department. Cat # Product Name Unit Size A31856 anti-oxphos Complex I 15 kda subunit, mouse IgG 1, monoclonal 17G µg A21359 anti-oxphos Complex I 17 kda subunit, mouse IgG 2b, monoclonal 21C11 *human mitochondrial reactivity* µg A31857 anti-oxphos Complex I 20 kda subunit, mouse IgG 1, monoclonal 20E9 *human mitochondrial reactivity* µg A21343 anti-oxphos Complex I 30 kda subunit, mouse IgG 1, monoclonal 3F9 *human mitochondrial reactivity* µg A21344 anti-oxphos Complex I 39 kda subunit, mouse IgG 1, monoclonal 20C11 *human mitochondrial reactivity* µg A21345 anti-oxphos Complex II 30 kda subunit, mouse IgG 2a, monoclonal 21A11 *human mitochondrial reactivity* µg A11142 anti-oxphos Complex II 70 kda subunit, mouse IgG 1, monoclonal 2E3 *human mitochondrial reactivity* µg A21361 anti-oxphos Complex III 10 kda subunit, mouse IgG 2a, monoclonal 1H µg A21362 anti-oxphos Complex III core 1 subunit, mouse IgG 1, monoclonal 16D10 *human mitochondrial reactivity* µg A11143 anti-oxphos Complex III core 2 subunit, mouse IgG 1, monoclonal 13G12 *human mitochondrial reactivity* µg A21346 anti-oxphos Complex III subunit FeS, mouse IgG 2b, monoclonal 5A5 *human mitochondrial reactivity* µg A6403 anti-oxphos Complex IV subunit I, mouse IgG 2a, monoclonal 1D6 (anti-cytochrome oxidase subunit I) A21296 anti-oxphos Complex IV subunit I, mouse IgG 2a, monoclonal 1D6, Alexa Fluor 488 conjugate (anti-cytochrome oxidase subunit I, Alexa Fluor 488 conjugate) *1 mg/ml* *human mitochondrial reactivity* µl A21297 anti-oxphos Complex IV subunit I, mouse IgG 2a, monoclonal 1D6, Alexa Fluor 594 conjugate (anti-cytochrome oxidase subunit I, Alexa Fluor 594 conjugate) *1 mg/ml* *human mitochondrial reactivity* µl A6404 anti-oxphos Complex IV subunit II, mouse IgG 2a, monoclonal 12C4 (anti-cytochrome oxidase subunit II) A21347 anti-oxphos Complex IV subunit IV, mouse IgG 2a, monoclonal 10G8 (anti-cytochrome oxidase subunit IV) A21348 anti-oxphos Complex IV subunit IV, mouse IgG 2a, monoclonal 20E8 (anti-cytochrome oxidase subunit IV) A21363 anti-oxphos Complex IV subunit Va, mouse IgG 2a, monoclonal 6E9 (anti-cytochrome oxidase subunit Va) A21349 anti-oxphos Complex IV subunit Vb, mouse IgG 2b, monoclonal 16H12 (anti-cytochrome oxidase subunit Vb) A21365 anti-oxphos Complex IV subunit VIa-L, mouse IgG 1, monoclonal 14A3 (anti-cytochrome oxidase subunit VIa-L) A21366 anti-oxphos Complex IV subunit VIb, mouse IgG 1, monoclonal 3F9 (anti-cytochrome oxidase subunit VIb) A6401 anti-oxphos Complex IV subunit VIc, mouse IgG 2b, monoclonal 3G5 (anti-cytochrome oxidase subunit VIc) A21367 anti-oxphos Complex IV subunit VIIa-H/L, mouse IgG 2a, monoclonal 6D7 (anti-cytochrome oxidase subunit VIIa-H/L) µg A21368 anti-oxphos Complex IV subunit VIIb, mouse IgG 1, monoclonal 2G7 (anti-cytochrome oxidase subunit VIIb) A21355 anti-oxphos Complex V inhibitor protein, mouse IgG 1, monoclonal 5E2 (anti-atp synthase IP; anti-f 1 -ATPase IP) A21351 anti-oxphos Complex V subunit, mouse IgG 1, monoclonal 3D5 (anti-atp synthase subunit ; anti-f 1 -ATPase subunit ) A21350 anti-oxphos Complex V subunit, mouse IgG 2b, monoclonal 7H10 (anti-atp synthase subunit ; anti-f 1 -ATPase subunit ) A21353 anti-oxphos Complex V subunit d, mouse IgG 2b, monoclonal 7F9 (anti-atp synthase subunit d; anti-f 1 -ATPase subunit d) A21354 anti-oxphos Complex V subunit OSCP, mouse IgG 1, monoclonal 4C11 (anti-atp synthase subunit OSCP; anti-f 1 -ATPase subunit OSCP) *human mitochondrial reactivity* µg A31855 anti-porin (human mitochondrial), mouse IgG 2b, monoclonal 31HL *1 mg/ml* µl B10710 BlockAid blocking solution *for use with microspheres* ml C24430 CoroNa Red chloride... 1 mg I36933 Image-iT FX signal enhancer ml P36930 ProLong Gold antifade reagent ml P36931 ProLong Gold antifade reagent with DAPI ml P36935 ProLong Gold antifade reagent with DAPI *special packaging*... 5 x 2 ml P36934 ProLong Gold antifade reagent *special packaging*... 5 x 2 ml

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