Chem 509 Signal Transduction Winter 2012

1. Chem 509 • Signal Transduction • Winter 2012 Lecture 5

2. 1. Two-hybrid assay. • Created by Song and Fields. • Concept adapted from the studies of Ptashne • Modular nature of proteins. • Companies now make kits for this

3. 2. IP Kinase activity assay • Assay employs immunoprecipitation technique. • Can use radiolabeled ATP or GTP • Can use Western blotting to detect phosphorylated substrate.

4. 3. FRET (Förster resonance energy transfer) • Depends on transfer of energy from one molecule to another. (1-10 nm) • Requires two proteins to be modified and then expressed inside cells. • cyan fluorescent protein and yellow fluorescent protein are commonly used. • Comment on green fluorescent protein. • Detect by microscopy-makes pretty images.

5. 4. Knockout mice • Powerful technique for determining the function of genes. • Assumes that human gene functions analogously in mice (not always true). P53 yes, • RB no. • Knockin mice, transgenic mice • Conditional knock out mice

8. 5. RNA interference (knockdown) • Good for tissue culture work. • Not easy to design the double stranded RNA to be used. • Delivery of the dsRNA into the cell can be technically difficult. • Sometimes plasmids expressing dsRNA can be delivered.

10. 6. Surface Plasmon Resonance (SPR) • Shows association and dissociation of large molecules (proteins) • Can calculate affinity constants and dissociation constants. • Requires expensive equipment.

11. Nelson and Cox, Lehninger Biochemistry

12. References Two hybrid assay: Fields S, Song O (1989). "A novel genetic system to detect protein-protein interactions". Nature 340 (6230): 245-6. FRET: ^ Förster T. Zwischenmolekulare Energiewanderung und Fluoreszenz, Ann. Physik 6(2):55, 1948. Green fluorescent protein: Prendergast F, Mann K (1978). "Chemical and physical properties of aequorin and the green fluorescent protein isolated from Aequorea forskålea". Biochemistry 17 (17): 3448-53. Knockout mice: Capecchi MR. Generating mice with targeted mutations. Nat Med. 2001;7:1086-90. RNA interference: Fire, A., Albertson, D., Harrison, S., and Moerman, D. (1991) Production of antisense RNA leads to effective and specific inhibition of gene expression in C. elegans muscle. Development 113:503-514. Surface Plasmon Resonance (SPR): Aslan K, Lakowicz JR, Geddes C. Plasmon light scattering in biology and medicine: new sensing approaches, visions and perspectives. Current Opinion in Chemical Biology 2005, 9:538–544. Chromatin Immunoprecipitation (ChIP): Collas P, Dahl JA., Chop it, ChIP it, check it: the current status of chromatin immunoprecipitation. Front Biosci. 2008, 13:929-43.