Binding Assay Data 1 Performed 6.21.2010
The first binding assay showed progressive binding through the rounds with a decrease at round 5 and an increase at round 7B. When restarting selection after a brief 2 week hiatus, it was discovered that the DNA in round 5 (1) had degraded into a short unusable strands and therefore, selection had to be restarted at round 3 and then progressed with parallel selections (A and B) per selection, per round. There is not a great amount of deviation from the average, which shows promise that the percentage binding is accurate. The green bar indicates the percentage of total binding species while the red bar indicates the total percentage of species that bind to things other than the protein.
Binding Assay 2 Performed 7.10.2010
This binding assay shows that with progressive rounds after round 7, there has been an increase in background binding, relative to the total binding. The assay for round 10 was not completed without problems due to an odd smear on the cartridge, despite multiple tries with different Storm Imagers and different cassettes. The cassettes and the screen were exposed for approximately 30 minutes each due to the high radioactivity of the P-32 used during the assay. Round 7 shows the most promise for the A round and will later be sequenced. When visualizing the cassette, there was such overexposure of the bottom where the nonbinders had washed out that it had essentially bleached the top part white. The pixel reader, however, can determine the differences of minute shades of pixels, so percentage binding can still be achieved. The original assay was supposed to be performed with rounds 7A, 8B, 8A, 10B, 10A, but due to the low yield after the etOH precipitation, it was impossible to continue. The top half of the cassette shows the binders for the assay with 1027 protein. The bottom half of the cassette shows that which was done without the 1027 protein. The blots in the top grid (1) cannot be seen, but based on the bottom grid (2). Grid 2 represents the washed away non binders. The darker blots on the bottom half of the grid show that more has washed out during the negative protein assay, which is promising. The green bars indicate percentage of total binding species and the red bars indicate percentage of total nonspecific binding species.
3 comments:
Please don't just post data without explaining it. What it means, what it tells you, what you did and what you plan to do, what the pool, target, conditions, round, etc are.
When you post it, make sure the data labels are accurate. The X axis for assay 2 doesn't have the correct round number. The actual dot blot does not describe what the boxes are.
I am doing that right now. I just wanted to see how the post would look. I loaded the wrong pictures on so it's all different.
In response to the statement about DNA degrading after 2 weeks, I don't think this is very likely. It's more likely you had an odd PCR product (i.e. smaller size) instead of DNA degradation.
Nice job with the selection. It looks like you're getting somewhere with it.
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