Cell Surface Targeting

1
Cell Surface Targeting

• 7/31/06

2
Last Time

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

3
Last week

• Introduced denaturation step, human thrombin
• No dice.

4
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.

5
3 new rays of hope (assays)

• Nitrocellulose filter plates
• Surface Plasmon Resonance
• Biacore 3000 at CGR
• Streptavidin-agarose beads

6
Surface Plasmon Resonance
7
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

1 2 3 4 5 6 7 8 9 10 11 12
Gel run for one hour
T35
T50
T20
S50
S20
S35
8
T20 secondary structure
T5
9
Running a gel for 2 hours

• 1 ladder
• 2 T20
• 3 T20 thrombin
• S20
• S20 thrombin
• T20S20
• T20S20 thrombin

1 2 3 4 5 6 7
10

• 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.

11
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