General+Meeting+Presentation+10-21-12

This presentation was a summary of screening the tumor cDNA library (no sequence information at this point yet), antibody purification efforts, linearization of plasmid for library, and first look at random hexamer library.

This will mostly be an update to the presentation given on General Meeting Presentation 7-25-12

This presentation will actually take place on 10-24-12.

presentation can be found here C:\kurt\storage\CIM Research Folder\kwhittem\Presentations\2012\General Meeting Presentation 10-24-12

10-24-12 Notes after presentation

It was clear after this presentation that Kathy, Stephen, and myself are not all on the same page together about what has been done and what should be done. Well, I suppose this makes sense since there has been much more communication between Kathy and I than Stephen. Here are some of the things that were brought up in the Q&A or during post meeting discussion that I can remember.

Stephen was wondering why I didn't discover SMC1 in the library. . . I was never actively searching for this, and I think it is unlikely to find this protein.

Stephen asked why everything was taking so long, and why I had not identified any sequences. I went back through the presentation, and showed some sequences I think I have identified such as the DEAD box protein. It turns out that Phil has done some research with the DEAD box protein.

I mentioned to Stephen that just the protein was put into the library, but Kathy reminded me afterward that I did put in a SMC1fs plasmid into some bacteria.

Tiger asked how I would make phage for human. I replied this could be difficult without access to a spleen. Kathy reminded me after the meeting that we can use PBMCs (peripheral blood monocytes). Humans are much larger than mice so it is easier to obtain PBMCs.

Krupa thinks there may be some issues with background with red and green channels.

Bart asked how the time of traditional panning compares to my pooling nitrocellulose method. Kathy thinks this would be a good thing to find out and possibly bring up in a committee meeting.

Bart mentioned that I should not refer to my fold change-p value plots as volcano plots but just as scatterplots like in flow cytometry since I don't have the other half.

Kathy is skeptical about whether the spots I've identified such as 31, 33, and 42 are real since the p values and fold changes are not great. I asked how I could further verify them. She mentioned possibly with RT-PCR.

I talked to Phil about the p values. He didn't have anything specific to say about how an appropriate p value could be determined when moving from the 3,000 spot array to a much smaller array from that one. However, he did say that he would think that the p values would get better as fewer clones were included into the spots. He also admitted that other factors could be at play (maybe more tumor proteins in the pool, differences in protein expression batches, differences in slide batches, etc.).

Phil thought it might be good to look at approximately how many proteins can typically be identified in this type of screen, and he recommended looking at the literature. He e-mailed me a list of literature that he has: Phage and tumor reading list from Phil 10-24-12

Kathy thinks it would be good to look at my sequences in detail to see if any of them are frameshifts.