Part Number Item MSFX X Detector Antibody 0.2 ml X HRP Label 0.2 ml Microplate strips (4-pack) 1

Transcription

1 Order: Web: Support: TECHNICAL DATA SHEET Complex I FlexPlex Module Catalog # MSFX-1 Rev.0 LOT #: APPLICATIONS: SPECIES CROSS- REACTIVITY: KIT COMPONENTS: Analysis of Complex I (NADH dehydrogenase, NADH ubiquinone oxidoreductase) quantity Human, bovine Part Number Item MSFX X Detector Antibody 0.2 ml X HRP Label 0.2 ml Microplate strips (4-pack) 1 STORAGE CONDITIONS: COUNTRY OF ORIGIN: Store all tubes and the microplate strips at 4 C. The kit is stable for 6 months. USA OVERVIEW: The Complex I FlexPlex Module (Catalog #MSFX-1) is used to determine the quantity of Complex I (E.C ) enzyme in a human or bovine sample. Complex I is immunocaptured with a monoclonal antibody within the wells and the quantity of protein is measured by adding a second Complex I-specific monoclonal antibody that is then labeled with horseradish peroxidase. This peroxidase changes the substrate from colorless to blue and the rate of color development is proportional to the amount of protein captured in the well, which can be monitored at 600 nm. Alternatively, the assay can be terminated at a user-defined time by the addition of 1N HCl (not supplied) and the assay performed as an end-point measurement at 450 nm. NOTE: The FlexPlex Starter Kit (MSFX-ST) is required for this assay.

2 SPECIFICITY/SENSITIVITY: The specificity of the Complex I FlexPlex Module (Catalog # MSFX-1) has been demonstrated by MALDI and LCMS mass spectrometry in combination with electrophoresis (1,2). ASSAY PERFORMANCE DATA MSFX-1 Range (min. 2x) background) HepG µg / well Avg. Intra-assay %CV (n=6) 5.4% Species cross-reactivity Cellular location Genetic origin Human, bovine Mitochondrial inner membrane Dual nuclear / mitochondrial (1) Schilling B. et al., Mol Cell Proteomics Jan;4(1): Epub 2004 Dec 10. Rapid purification and mass spectrometric characterization of mitochondrial NADH dehydrogenase (Complex I) from rodent brain and a dopaminergic neuronal cell line. (2) Murray J. et al., J. Biol. Chem Apr 18;278(16): The subunit composition of the human NADH dehydrogenase obtained by rapid one-step immunopurification.

3 EXAMPLE DATA 1: FlexPlex Modules MSFX-1, 2, 3, 4, 5 are assays designed to analyze the relative levels of the 5 OXPHOS complexes between samples. An experiment to demonstrate this is shown below in which fibroblast cell lines derived from patients with mitochondrial genetic disease are compared to a control fibroblast cell line (newborn human dermal fibroblast). EXPERIMENTAL DATA 1 FIBROBLASTS Fibroblasts extract loaded per well Signal/background (control) Reproducibility (%CV 6 wells) MSFX-1 [Complex I] 50 µg MSFX-2 [Complex II] 50 µg MSFX-3 [Complex III] 12.5 µg MSFX-4 [Complex IV] 50 µg MSFX-5 [Complex V] 12.5 µg MSFX-7 [Frataxin] 25 µg MSFX-6 [GAPDH] µg The raw signals can be compared between samples or in the example shown above the signals can be normalized to GAPDH. After normalization the patient cell line 1 has 50-60% reduction in assembly of complex I, II, and III but also has a previously unmeasured 85% reduction in frataxin signals. While the patient cell line 2 has a specific 95% reduction in Complex IV levels when compared to the control cell line (newborn human dermal fibroblast).

4 Relative Signal/GAPDH EXAMPLE DATA 2: Mitochondrial DNA disruption or loss results in abnormal assembly of OXPHOS components. An experiment to demonstrate this is shown below in which 143B osteosarcoma cell lines depleted in mitochondrial DNA (Rho0 cells) are compared to an unmodified control 143B osteosarcoma cell line. EXPERIMENTAL DATA 2 Rho0 cells 143B extract loaded per well Signal/background (control) Reproducibility (%CV 4 wells) MSFX-1 [Complex I] 50 µg MSFX-2 [Complex II] 50 µg MSFX-3 [Complex III] 12.5 µg MSFX-4 [Complex IV] 50 µg MSFX-5 [Complex V] 12.5 µg MSFX-7 [Frataxin] 25 µg MSFX-6 [GAPDH] µg #1 -Complex I #2 -Complex II #3 -Complex III #4 -Complex IV #5 -ATP synthase #7 -Frataxin #6 -GAPDH Control 143B cell line Clonal Rho0 143B cell line After normalization of all raw signal rates to GAPDH the Rho0 cell line (lacking mitochondrial DNA) has a >95% reduction in complex I, III, IV. Interestingly an 82% drop in assembled Complex II is also identified and a 79% decrease in frataxin both of which are presumably due to a disruption in mitochondrial membrane organization.

5 EXAMPLE DATA 3: Drug induced mitochondrial toxicity is a well recognized phenomenon. Two examples of which are the inhibition of mitochondrial DNA replication by nucleoside analogs reverse transcriptase inhibitors (NARTI) and the inhibition of mitochondrial protein expression by inhibitors of the mitochondrial ribosome (e.g. antibiotics). The effect of a drug from each of these classes on mitochondrial OXPHOS enzyme assembly is shown below. Human HepG2 cells were cultured in each drug for 6 days to ensure a significant effect on mitochondrial DNA replication and mitochondrial protein translation, respectively. The NARTI ddc (Zalcitabine) reduced mitochondrial DNA levels and hence mitochondrial protein expression. As a consequence the assembly of Complexes I, III and IV were severely affected. Note that loss of the two small mitochondrial DNA encoded subunits of Complex V does not affect overall assembly. Interestingly an increase in Complex II was induced as a consequence of I, III, IV loss possibly to maintain mitochondrial function. The antibiotic chloramphenicol inhibited mitochondrial protein translation and assembly of Complexes I and IV but had no effect on Complex III.