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Cell Surface Targeting: The week of waiting

Cell Surface Targeting: The week of waiting. 8/14/06. Adaptamers. T20. A20. S20. T35. A35. S35. T50. Link-aptamer. A50. S50. Outline. Thrombin aptamer/thrombin beads A20 experiment Magnetic beads. T0: thrombin aptamer (15 bp single stranded) SB: streptavidin beads

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Cell Surface Targeting: The week of waiting

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  1. Cell Surface Targeting: The week of waiting 8/14/06

  2. Adaptamers T20 A20 S20 T35 A35 S35 T50 Link-aptamer A50 S50

  3. Outline • Thrombin aptamer/thrombin beads • A20 experiment • Magnetic beads

  4. T0: thrombin aptamer (15 bp single stranded) SB: streptavidin beads TB: thrombin beads Thrombin aptamer 1 2 3 4 5 6 7 8 9 1: dsDNA ladder 2: 20 pmol T0 3: 10 uL TB + 80 pmol T0 4: 25 uL TB + 80 pmol T0 5: 50 uL TB + 80 pmol T0 6: 100 uL TB + 80 pmol T0 7: 150 uL TB + 80 pmol T0 8: 150 uL TB + S5 (50bp, single stranded oligo) 9: 150uL SB + 80 pmol T0 50 40 30 20 10 Above: Elutions with thrombin Below: Washes with buffer 50 40 30 20 10

  5. A20 T20 A20 experiment S20 • Incubated adaptamer A20 and thrombin with streptavidin beads; washes and elutions using free streptavidin were collected. • As controls, streptavidin beads + thrombin were also incubated with T20 or S20, and trypsin beads + thrombin were incubated with A20.

  6. A20 Experiment: DNA 1 2 3 4 5 6 7 8 9 10 11 1: ladder 2: 20 pmol T20 3: 20 pmol S20 4: 20 pmol A20 5-8: SB + A20 + varying concentrations of thrombin 9: SB + T20 + 320 pmol thrombin 10: SB + S20 + 320 pmol thrombin 11: TrB + A20 + 320 pmol thrombin 100 uL beads, 160 pmol oligo in experimental lanes 50 40 30 20 10 Above: Elutions with streptavidin Below: Washes with buffer 50 40 30 20 T0: thrombin aptamer (15 bp single stranded) SB: streptavidin beads TrB: trypsin beads 10

  7. A20 Experiment: Protein 1: ladder 3: 40 pmol thrombin 4:SB + A20 + 40 pmol thrombin 5:SB + A20 + 80 pmol thrombin 6:SB + A20 + 160 pmol thrombin 7:SB + A20 + 320 pmol thrombin 8: SB + T20 + 320 pmol thrombin 9: SB + S20 + 320 pmol thrombin 10: TrB + A20 + 320 pmol thrombin 100 uL beads, 160 pmol oligo in experimental lanes 1 2 3 4 5 6 7 8 9 10 “250” “148” “98” “64” “250” “148” Above: Elutions with streptavidin Below: Washes with buffer “98” T0: thrombin aptamer (15 bp single stranded) SB: streptavidin beads TrB: trypsin beads

  8. Elutions Pt. 2: Silver Staining 7:SB + A20 + 320 pmol thrombin 8: SB + T20 + 320 pmol thrombin 9: SB + S20 + 320 pmol thrombin 10: TrB + A20 + 320 pmol thrombin 100 uL beads, 160 pmol oligo in experimental lanes 1: ladder 3: 40 pmol thrombin 4:SB + A20 + 40 pmol thrombin 5:SB + A20 + 80 pmol thrombin 6:SB + A20 + 160 pmol thrombin T0: thrombin aptamer (15 bp single stranded) SB: streptavidin beads TrB: trypsin beads 1 2 3 4 5 6 7 8 9 10

  9. Elutions Pt. 3: SDS-PAGE 1 2 3 4 5 6 7 8 9 10 250 1: ladder 2: 20 pmol thrombin 3: 20 pmol streptavidin 4:SB + A20 + 40 pmol thrombin 5:SB + A20 + 80 pmol thrombin 6:SB + A20 + 160 pmol thrombin 7:SB + A20 + 320 pmol thrombin 8: SB + T20 + 320 pmol thrombin 9: SB + S20 + 320 pmol thrombin 10: TrB + A20 + 320 pmol thrombin 100 uL beads, 160 pmol oligo in experimental lanes 148 98 64 50 36 22 16 6 4 T0: thrombin aptamer (15 bp single stranded) SB: streptavidin beads TrB: trypsin beads

  10. L: ladder 1:SB + A20 + 40 pmol thrombin T: thrombin 2:SB + A20 + 80 pmol thrombin S: streptavidin 3:SB + A20 + 160 pmol thrombin 4:SB + A20 + 320 pmol thrombin 5: SB + T20 + 320 pmol thrombin 6: SB + S20 + 320 pmol thrombin 7: TrB + A20 + 320 pmol thrombin T0: thrombin aptamer (15 bp single stranded) SB: streptavidin beads TrB: trypsin beads L T 1 2 3 4 5 6 7 L T S 1 2 3 4 5 6 7 “250” 250 “148” 148 “98” 98 “64” 64 50 “250” 36 “148” 22 “98” 16 “64” 6 4 12% Native PAGE 12% SDS-PAGE

  11. By the way.. 1: Seeblue plus2 ladder 2: BSA 3: human thrombin (same used in experiment) 4: Sigma human thrombin 5: Sigma bovine thrombin 6: streptavidin 250 148 98 64 50 36 16 6 4

  12. Magnetic Beads • Received streptavidin magnetic beads • Incubated magnetic beads with A50 or S0, then added thrombin beads. • Magnetic and thrombin beads clump together!

  13. Streptavidin beads + Control: streptavidin beads+ thrombin beads +A50 thrombin beads + S0

  14. Control: beads + S0 Beads + A50

  15. Streptavidin beads + Control: streptavidin beads + thrombin beads +A50 thrombin beads + S0

  16. Streptavidin beads + Control: streptavidin beads + thrombin beads +A50 thrombin beads + S0

  17. Streptavidin beads + Control: streptavidin beads + thrombin beads +A50 thrombin beads + S0

  18. Streptavidin beads + Control: streptavidin beads + thrombin beads +A50 thrombin beads + S0

  19. Streptavidin beads + Control: streptavidin beads + thrombin beads +A50 thrombin beads + S0

  20. Streptavidin beads + A50 Control: Streptavidin beads + S0

  21. Streptavidin beads + Control: streptavidin beads + thrombin beads +A50 thrombin beads + S0

  22. Streptavidin beads + Control: streptavidin beads + thrombin beads +A50 thrombin beads + S0

  23. Next: • Get new adaptamers! • Wash streptavidin beads; run on gel. • Retry bead assay with new adaptamers. • Try mixing streptavidin agarose and magnetic beads with biotinylated oligos. • At the very least, run gel which shows that adaptamers can bind thrombin and streptavidin beads separately.

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