- The A20 experiment
Question: Is A20.1 able to bind both of its targets at once?
Setup: We will add a constant amount of A20 to a constant volume of streptavidin beads, then incubate with varying amounts of thrombin. After a few washes, we will elute whatever is bound to the beads with free streptavidin. Controls will be replacement of A20 with S20 or T20 (nonspecific interaction of thrombin with DNA for former, binding of thrombin to beads via T20 alone in latter) and replacement of streptavidin beads with trypsin beads (testing binding to agarose).
Specific setup: A20 was prepared by mixing equal molar solutions of S20 and T20, heating to 95 C and cooling slowly to room temperature for 20 minutes. Streptavidin beads were washed three times with Bittker buffer and resuspended in an equal volume of Bittker buffer. 100 uL streptavidin or trypsin beads were combined with 160 pmol A20, S20, or T20 to a total volume of 120 uL and incubated for 30 minutes on a shaker. Varying concentrations of thrombin were added and shook for 30 minutes more. Solutions were then washed three times in Bittker buffer; first wash was collected. Bound substrates were then eluted by incubating with 100 uL 50% w/v streptavidin for 1 hour on a shaker. Solutions were then centrifuged and elutions collected. 8 uL of solutions + 2uL 5X TBE Hi Density Loading Buffer were loaded onto 4-20% pre-cast polyacrylamide gels in TBE buffer and run for 30 minutes. 9 uL solutions + 1 uL 10X Tris-glycine loading buffer were run on 12% pre-cast polyacrylamide gels in Tris-glycine buffer. Duplicate samples of washes and elutions were stained with either Coomassie blue or Ethidium Bromide.
Elutions: Increasing Coomassie staining as the concentration of thrombin increases; no thrombin in any control lane.
Washes: Decreasing Coomassie staining as the concentration of thrombin increases; strong bands in control lanes.
Elutions: Greater intensity for lanes with A20, S20
Washes: Greater intensity for lanes with T20, trypsin beads.
Background: From the past experiments with thrombin and streptavidin beads, we know that following a wash, more S0 remains on streptavidin beads than T0 on thrombin beads when the same concentrations of aptamer and streptavidin is used. A number of factors may have contributed to this observation, among them the fact that bovine rather than human thrombin is conjugated to the agarose. Therefore we will use streptavidin rather than thrombin beads as a base. Using either type of beads, a large fraction of even pure aptamer is lost during the wash; therefore it make sense to try rather large amounts of beads, adaptamer, and thrombin concentrations.
Conditions labeled as follows:
SB: streptavidin beads TrB: trypsin beads
1: 100 uL SB + 160 pmol A20 + 40 pmol thrombin 2: 100 uL SB + 160 pmol A20 + 80 pmol thrombin 3: 100 uL SB + 160 pmol A20 + 160 pmol thrombin 4: 100 uL SB + 160 pmol A20 + 320 pmol thrombin 5: 100 uL SB + 160 pmol T20 + 320 pmol thrombin 6: 100 uL SB + 160 pmol S20 + 320 pmol thrombin 7: 100 uL TB + 160 pmol A20 + 320 pmol thrombin
|2||20 pmol T20|
|3||20 pmol S20|
|4||20 pmol A20|
|3||40 pmol thrombin|
A20 binds well to streptavidin. Whether thrombin does is still up to question. The streptavidin found in the elution makes it difficult to detect trace amounts of thrombin (and hopefully there is some). However, we'll take advantage of the fact that silver staining does not stain streptavidin well in another attempt to detect thrombin tomorrow.