Over the past two months, I have completed 6 rounds of filterbased selection against Dengue Virus Envelope Protein(DENG) and have performed a binding assay on Rounds 1, 4, 5, and 6. I am currently repeating Round 5. To decrease the size of my posts I am providing links to the images of my cycle course gels. For more information on the target click here.
Round 1 ccPCR Gel: Cycle 15 showed the greatest amplification of DNA without over amplification.
Round 3 ccPCR Gel: Cycle 12 showed the greatest amplification without over amplification. Plus and Minus protein showed the same amplification patter. Since this is Round 3 this is not overly concerning. However, if this continues into later rounds, it may indicate a substantial presence of background binders.
Round 4 ccPCR Gel: Cycle 12 showed the greatest amplification without over amplification. Plus protein first showed amplification at Cycle 9. Minus protein first showed amplification at Cycle 12. This suggests that binding species are beginning to dominate the pool.
Round 6 ccPCR Gel: Cycle 9 showed the greatest amplification without over amplification. Both Minus Elutions and the Plus DENG Elution showed the same amplification pattern. This unfortunately suggests the continued presence of background binders despite the increase in wash volumes and negative selection.
Round 6B ccPCR Gel: tRNAs and decreased protein concentration were used for this round. Cycle 12 showed the greatest amplification without over amplification. Plus protein amplified later than minus suggesting a strong presence of background binding.
Round 7B ccPCR Gel:Cycle 9 showed the greatest amplification without over amplification. Both Minus Elutions and the Plus DENG Elution showed the same amplification pattern. This unfortunately suggests the continued presence of background binders despite the addition of tRNAs.
Binding Assay R1, R4, R5, R6: A binding assay was performed on Rounds 1, 4, 5, and 6 RNA. The image below shows the results of the assay. This assay shows promising results. The there was a gradual increase in binding of the pool to about 5% by R5, without a substantial presence of background binding, approximately 2%, until Round 6. The binding of the pool seems to level off between Rounds 5 and 6. This combined with the presence of background binding in R6 means that I will go back to R5 and continue through selection. To increase the binding of the pool, I will drop my protein concentration to 100pmol and select for more specific binders. To prevent a rise in background binding, I will use tRNAs to block the nonspecific binding sites of the protein and the filter. After going back to R5, I will carry the pool through two more rounds of selection before performing another binding assay.
A) Shows the developed image of the phosphor plate exposed to the filters containing the radioactive RNA. B) Shows the % binding of the assayed rounds.
Binding Assay R6, R6B, R7B: A binding assay was performed on R6, R6B, and R7B. The percent binding results can be seen below. R6B and R7B are a continuation from R5 with added tRNAs and decreased protein concentration. The increase in background binding suggests that the tRNAs did not effectively block general binding sites. Additionally, the decreased protein concentration most likely favored background binders over protein binders. This assay also raises some concern as to the accuracy of the first assay, since R6 results differ between the two assays.
Shows the % binding of the assayed rounds
3 comments:
So far so good! Make sure to label this as the elution. I had dropbox loading issues so I couldn't check, but are you doing bead or filter based slx? If you're doing bead, initially you might want to show some W3 data. Its good to see if your E amplifies sooner than weaker binding wash species.
I'm wondering why your round 2 cycle 15 has two bands? Any ideas?
To holli's question, I am doing filter based slx. As for the question about the two bands, I am not entirely sure. A sequence could have been lengthened by a strange primer effect. This particular sequence could have been replicated more readily by Taq. This could have lead to one particular length sequence that rose to a noticeable concentration.
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