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There is another top address in Darmstadt! AppliChem CheLuminate AppliChem © 2010 1/14 There is another top address in Darmstadt! CheLuminate • Chemiluminescence detection kits • replaces radioactive assays • extremely sensitive AppliChem © 2010 2/14 There is another top address in Darmstadt! CheLuminate • Horseradish Peroxidase-driven • Luminol-based • plus enhancer • New: plus nucleophilic acylation catalyst AppliChem © 2010 3/14 There is another top address in Darmstadt! CheLuminate • Chemiluminescence detection kits o for ELISA CheLuminate-HRP ELISA FemtoDetect CheLuminate-HRP ELISA FemtoDetect Plus o for highest sensitivity CheLuminate-HRP FemtoDetect CheLuminate-HRP FemtoDetect Plus CheLuminate-HRP PicoDetect Extended o for standard applications CheLuminate-HRP PicoDetect (with phenolic enhancer) AppliChem © 2010 4/14 There is another top address in Darmstadt! CheLuminate • AppliChem's products: 10-12 10-12 10-14 10-15 AppliChem © 2010 5/14 There is another top address in Darmstadt! CheLuminate • Sensitivity and stability Comparison of CheLuminate PicoDetect Extended with a competitor (B) AppliChem © 2010 6/14 There is another top address in Darmstadt! CheLuminate • AppliChem CheLuminate – Sales arguments o competitive price o highest sensitivity available – from pico (10-12) to femto (10-15) range o extremely high stability of reagents – up to 24 h signal duration o compatible with most detection systems / instruments AppliChem © 2010 7/14 There is another top address in Darmstadt! CheLuminate • Competitors o Pierce (Thermo Scientific) AppliChem © 2010 Pierce ECL, SuperSignal™ West Pico, SuperSignal™ West Femto o Millipore Immobilon™ Western HRP o GE healthcare ECL™ 8/14 There is another top address in Darmstadt! CheLuminate • Literature AppliChem © 2010 9/14 There is another top address in Darmstadt! Bio-/Chemiluminescence • Luciferase System o Luciferase is one of the most sesnitive reporter in gene expression studies; no radioactivity Zu bearbeiten o No cellular background activity of luciferase in most assay systems o Easy to perform o Linear activity over a wide range APMBT AppliChem © 2010 10/14 There is another top address in Darmstadt! Bio-/Chemiluminescence • Luciferase System with APMBT o Luciferase from Photinus pyralis (Pluc) o Luciferase from Renilla reniformis (Rluc) Zu bearbeiten o application of both luciferases in the same assay possible: internal control or investigation of two transcription signals o first, measurement of Pluc, then Rluc under Rluc assay conditions, Pluc inactive, because APMBT quenches Pluc o This assay is compatible with protein assays!* o o o AppliChem © 2010 APMBT = 2-(4-Aminophenyl)-6-methylbenzothiazole) * Reference: Hampf, M. & Gossen, M. (2006) Anal. Biochem. 356, 94-99 comparable to Promega‘s DualGlow 11/14 There is another top address in Darmstadt! Bio-/Chemiluminescence • BCA o Simple, sensitive photometric assay for proteins. o Faster and easier than Lowry assay. o With greater tolerance to interference from non-ionic detergents, Zu bearbeiten o and simple buffer salts. More stable at alkaline pH. o o o o AppliChem © 2010 The principle of the bicinchoninic acid (BCA) assay is similar to the Lowry procedure, in that both rely on the formation of a Cu2+-protein complex under alkaline condition followed by reduction of the Cu2+ to Cu1+. The amount of reduction is proportional to the protein present. In the second step, BCA forms a complex with Cu1+, which is purple colored and is detectable at 562 nm. Protein + Cu2+ => Cu1+ + BCA => Cu1+-BCA complex market leader probably Pierce 12/14 There is another top address in Darmstadt! Bio-/Chemiluminescence • Overview o substrates & inducers for e.g. Cloning Blue/White Selection: X-Gal / IPTG e.g. Histology Alkaline Phosphatase Detection: the detection of • genes & proteins, • enzyme activities Zu bearbeiten BCIP / NBT Diformazan e.g. Western Blots Horseradish Peroxidase Detection: H2O2 Luminol CheLuminate-HRP Light e.g. Reporter Genes Firefly Luciferase Detection: ATP Luciferin AppliChem © 2010 Light 13/14