IGEM:Harvard/2006/Adaptamers/Notebook/2006-8-18

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8/18

Detection of thrombin binding to streptavidin using antibodies (cont'd)

Refrigerated solutions were diluted to 50 uL total volume and solutions entered into a plate reader. Plate was first scanned at high intensity and then at low intensity.

Well Condition
B2 A35
B4 A15
B6 NT
B8 1S
B10DS
B12 JB


  • High Intensity

Image:cst818solutionsHigh.jpg

  • Low Intensity

Image:cst818solutionsLow.jpg

  • Values
Condition Value
A35 3087.35
A15 3104.84
NT 489.97
1S 2097.13
DS 4083.89
JB 91.56

Image:cst818intensitygraph.jpg

Both A35 and A15 proved their ability to bind streptavidin and thrombin. Applying secondary and primary antibodies weakened the signal (1S): whatever adaptamers or thrombin remain bound thanks to fewer washes must be insufficient to counter the entrapment of secondary antibodies by unbound primary antibodies. As one might expect, doubling the amount of thrombin significantly increased the signal observed (DS).

Western blot (cont'd)

After protein was transferred to nitrocellulose, the membrane was stained with Ponceau Red and blocked for 2.5 hours. The membrane was then incubated with 1:500 goat anti-thrombin antibody for 90 minutes, washed 3X in PBS/.1% Tween, then incubated with 1:5000 rabbit anti-goat secondary antibody for 30 minutes and washed 3X in PBS before being imaged.

Ponceau Red:

Lane Condition
1SeeBlue Plus2 ladder
240 pmol thrombin
3A20 W
4A20 E
52X W
62X E
7A50 W
8A50 E
9ND W
10ND E
11TB W
12TB E

Image:cst818ponceaublot.jpg

Lane Condition
1SeeBlue Plus2 ladder
240 pmol thrombin
3A20 W
4A20 E
52X W
62X E
7A50 W
8A50 E
9ND W
10ND E
11TB W
12TB E

Image:cst818Western.jpg

The Western blot confirmed that A20 and A50 are also able to link streptavidin and thrombin. This experiment was very similiar to the one we conducted on 8/9. Using Ponceau Red, a less sensitive dye than Coomassie blue, we were still able to see thrombin in the blot. Either more was bound because we kept the volumes down and didn't have to use the SpeedVac to concentrate our solutions, or the new adaptamers are simply better designed. The degree of performance change suggests the latter.

All in all, an exciting day: two assays (although related) have verified the functionality of the adaptamers, and moreover, both are quantifiable. Special thanks to Alain who began the blocking reaction while I slept and made sure that we obtained good images.

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