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ETFA shRNA (h) Lentiviral Particles: sc-62259-V BACKGROUND APPLICATIONS ETFA (electron-transfer-flavoprotein, α polypeptide), also known as EMA, GA2 ETFA shRNA (h) Lentiviral Particles is recommended for the inhibition of or MADD, is a 333 amino acid protein that localizes to the mitochondrial ma- ETFA expression in human cells. trix and belongs to the ETF α-subunit/fixB family. Existing as a heterodimer with ETFB, ETFA uses FAD as a cofactor and serves as a specific electron ac- SUPPORT REAGENTS ceptor for several dehydrogenases, effectively transferring electrons to the Control shRNA Lentiviral Particles: sc-108080. Available as 200 µl frozen mitochondrial respiratory chain via ETF-ubiquinone oxidoreductase. Defects viral stock containing 1.0 x 10 6 infectious units of virus (IFU); contains an in the gene encoding ETFA are the cause of glutaric aciduria type IIA (GAIIA), shRNA construct encoding a scrambled sequence that will not lead to the a condition that is characterized by the excretion of lactic, ethylmalonic, bu- specific degradation of any known cellular mRNA. tyric, isobutyric, 2-methyl-butyric, glutaric and isovaleric acids. The gene en- coding ETFA maps to human chromosome 15, which houses over 700 genes GENE EXPRESSION MONITORING and comprises nearly 3% of the human genome. ETFA (H-8): sc-365982 is recommended as a control antibody for monitoring REFERENCES of ETFA gene expression knockdown by Western Blotting (starting dilution 1:200, dilution range 1:100-1:1000) or immunofluorescence (starting dilution 1. Frerman, F.E. 1988. Acyl-CoA dehydrogenases, electron transfer flavopro tein 1:50, dilution range 1:50-1:500). and electron transfer flavoprotein dehydrogenase. Biochem. Soc. Trans. 16: 416-418. To ensure optimal results, the following support (secondary) reagents are recommended: 1) Western Blotting: use goat anti-mouse IgM-HRP: sc-2064 2. Indo, Y., et al. 1991. Molecular characterization of variant α-subunit of elec - (dilution range: 1:500-1:5,000), TBS Blotto A Blocking Reagent: sc-2333 and tron transfer flavoprotein in three patients with glutaric acidemia type II— Western Blotting Luminol Reagent: sc-2048. 2) Immunofluorescence: use goat and identification of glycine substitution for valine-157 in the sequence of anti-mouse IgM-FITC: sc-2082 (dilution range: 1:100-1:400) or goat anti- mouse the precursor, producing an unstable mature protein in a patient. Am. J. IgM-TR: sc-2983 (dilution range: 1:100-1:400) with UltraCruz™ Mounting Hum. Genet. 49: 575-580. Medium: sc-24941. 3. Freneaux, E., et al. 1992. Glutaric acidemia type II. Heterogeneity in β-oxi - dation flux, polypeptide synthesis, and complementary DNA mutations in RT-PCR REAGENTS the α subunit of electron transfer flavoprotein in eight patients. J. Clin. Semi-quantitative RT-PCR may be performed to monitor ETFA gene expres sion Invest. 90: 1679-1686. knockdown using RT-PCR Primer: ETFA (h)-PR: sc-62259-PR (20 µl). Annealing temperature for the primers should be 55-60° C and the extension tempera - CHROMOSOMAL LOCATION ture should be 68-72° C. Genetic locus: ETFA (human) mapping to 15q24.2. BIOSAFETY PRODUCT Lentiviral particles can be employed in standard Biosafety Level 2 tissue ETFA shRNA (h) Lentiviral Particles is a pool of concentrated, transduction- culture facilities (and should be treated with the same level of caution as ready viral particles containing 3 target-specific constructs that encode with any other potentially infectious reagent). Lentiviral particles are repli- 19-25 nt (plus hairpin) shRNA designed to knock down gene expression. cation-incompetent and are designed to self-inactivate after transduction Each vial contains 200 µl frozen stock containing 1.0 x 10 6 infectious units and integration of shRNA constructs into genomic DNA of target cells. of virus (IFU) in Dulbecco’s Modified Eagle’s Medium with 25 mM HEPES pH 7.3. Suitable for 10-20 transductions. Also see ETFA siRNA (h): sc-62259 RESEARCH USE and ETFA shRNA Plasmid (h): sc-62259-SH as alternate gene silencing The purchase of this product conveys to the buyer the nontransferable right prod ucts. to use the purchased amount of the product and all replicates and derivatives for research purposes conducted by the buyer in his laboratory only (whether STORAGE the buyer is an academic or for-profit entity). The buyer cannot sell or other - Store lentiviral particles at -80º C. Stable for at least one year from the wise transfer (a) this product (b) its components or (c) materials made using date of shipment. Once thawed, particles can be stored at 4º C for up to this product or its components to a third party, or otherwise use this product one week. Avoid repeated freeze thaw cycles. or its components or materials made using this product or its components for Commercial Purposes. PROTOCOLS See our web site at www.scbt.com or our catalog for detailed protocols and support products. Santa Cruz Biotechnology, Inc. 1.800.457.3801 831.457.3800 fax 831.457.3801 Europe +00800 4573 8000 49 6221 4503 0 www.scbt.com.