Cell Surface Targeting

1
Cell Surface Targeting

• 8/21/06

2
Adaptamers
A15
T20
A20
S20
T35
A35
S35
T50
Link-aptamer
A50
S50
3
Outline

• Protein coupling to beads
• The new adaptamers
• Fluorescence Assay
• Western Blot
• New plans

4
Protein Coupling to Beads

• Coupled aminomethylfluorescein to carboxylated
  polystyrene beads

5
Coupled thrombin bead performance
L 1 2 3
4 5 6
50
40
30
20
10
6
New adaptamer coupling
L 1 2 3 4 5
6 7 8 9
100
50
40
30
20
10
7
New Assay to detect thrombin
Magnetic streptavidin beads
thrombin
Anti-thrombin 1o antibody
Wash
Shake
Shake
Wash
Secondary antibody
adaptamer
Shake
Infrared imager
Wash
Plate
Li-Cor
8
Success!
General setup Streptavidin beads A35 thrombin
Adaptamer A15
Control no thrombin
Primary and secondary antibodies added together
2X thrombin
Control beads lack streptavidin
9
Quantitative results
10
Western Blot
Agarose streptavidin beads
thrombin
Wash
Shake
Elute with free streptavidin
adaptamer
SDS-PAGE
Infrared imager
Blot, apply antibodies
Invitrogen
Li-Cor
11
Conditions

• General setup Streptavidin agarose beads A20
  thrombin
• Other conditions A50 instead of A20, double the
  amount of A20, thrombin
• Controls No adaptamer added thrombin agarose
  beads instead of streptavidin beads

12
DNA
L 1 2 3 4
5 6 7 8 9
10
100
50
40
30
20
10
A20 general setup Streptavidin agarose beads
A20 thrombin
A50 A50 instead of A20 ND No DNA added TB
Thrombin beads replace streptavidin beads
2X 2X thrombin, A20
13
Protein
L 1 2 3 4 5
6 7 8 9 10 11
36
16
6
A20 general setup Streptavidin
agarose beads A20 thrombin
2X 2X thrombin, A20 A50 A50 instead of A20 ND
No DNA added TB Thrombin beads replace
streptavidin beads
14
Ponceau Red
L 1 2 3 4 5
6 7 8 9 10 11
36
22
16
6
A20 general setup Streptavidin
agarose beads A20 thrombin
2X 2X thrombin, A20 A50 A50 instead of A20 ND
No DNA added TB Thrombin beads replace
streptavidin beads
15
2 weeks for the following

• New adaptamer that binds a cell surface
• Interchangeable parts proof of concept
• DNA candidates glioblastoma cells (tenascin-C),
  red blood cells, leukocytes (L-selectin)
• Leukocyte-thrombin?
• RNA candidates possible, too.
• Vary the linking region
• Strand displacement