Cell Surface Targeting 7/31/06

Last Time Identified two potential problems with our procedure –Use human thrombin, not bovine! –Denature aptamers!

Last week Introduced denaturation step, human thrombin No dice.

Liu lab Got in contact with Liu lab. “Gel shifts require that the off-rate (dissociation rate) be somewhat slow compared with the time scale of the electrophoresis and under the conditions of electrophoresis, and can be quite finicky.”

3 new rays of hope (assays) Nitrocellulose filter plates Surface Plasmon Resonance: Biacore 3000 at CGR Streptavidin-agarose beads

Surface Plasmon Resonance

Adaptamer subunit interaction 1: ladder 2: T20 3: S20 4: T20 + S20 5: T20 + S20 (denatured) 6: T35 7: S35 8: T35 + S35 9: T35 + S35 (denatured) 10: T50 11: S50 12: T50 + S50 (denatured) T oligos contain thrombin aptamer sequence S oligos contain streptavidin aptamer sequence Gel run for one hour T20 T35T50 S20S35 S50

T20 secondary structure T5

Running a gel for 2 hours 1: ladder 2: T20 3: T20 + thrombin S20 S20 + thrombin T20+S20 T20+S20+ thrombin

“Gel shifts require that the off-rate (dissociation rate) be somewhat slow compared with the time scale of the electrophoresis and under the conditions of electrophoresis, and can be quite finicky.”

Next Follow-up experiment: reattempt last experiment running gel for 1 hour Visit the Liu lab Talk with CGR Run experiments with streptavidin-agarose beads Design new DNA-DNA interactions to try