Cell Surface Targeting - PowerPoint PPT Presentation

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Cell Surface Targeting

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Last Time. Identified two potential problems with our procedure. Use human thrombin, not bovine! ... agarose beads. Design new DNA-DNA interactions to ... – PowerPoint PPT presentation

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Title: 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
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