Tumor+vs+normal+on+Single+Clones+8-27-12

Tumor vs normal on Single Clones 8-27-12

9-7-12 applied tumor and naïve sera to selected 16E3 tumor lysate. 1:500 dilution sera. Direct labeled 1 nM secondary used. 10 identical spots for each spot type were printed right next to eachother in this experiment. After this experiment, I decided to just print duplicates spaced far apart from one another. I also wanted to include plain e coli lysate in the future (not induced PUC19 lysate as was used in this experiment.) I also wanted to use 1:100 concentration of sera in the future.

actual experiment on 9-7-12

For this experiment I plan to print protein lysate from single colonies (see 8-26-12 Plasmids for verification) onto nitrocellulose arrays. These arrays will also have plain e coli lysate (actually it is induced PUC19 lysate) printed onto the array as a negative control. The arrays will be probed with naive sera and tumor sera.

9-6-12 Johnny printed the slides Gal file found here: "C:\kurt\storage\CIM Research Folder\DR\2012\9-6-12\array\kurt_4x4_090612_gal.gal"

also see How to get good results with lysate on nitrocellulose slides

experiment performed on 9-7-12 sera applied to array for overnight incubation on 9-7-12 secondary added and slides scanned on 9-8-12

Results found here "C:\kurt\storage\CIM Research Folder\DR\2012\9-8-12\nv-tmr-lst-slides\results 9-8-12.xlsx" Summary here "C:\kurt\storage\CIM Research Folder\DR\2012\9-17-12\Summary of 9-8-12 Experiment.docx"

I could look at this data further, but from what I see so far it looks like sample 42 (7F12 plasmid (see 8-26-12 Plasmids for verification)) is the highest compared with naive. I should see if this sequence is in frame and see how far it reads before reaching a stop codon in the plasmid.

Also, I may have done myself a disservice by spotting 10 identical spots right next to one another on the array. If there is a small amount of antibody then it may become spread out across all of the protein in these 10 adjacent spots making it harder to detect bright signals. I may want to print the spots in duplicates and have them far apart on the array. Additionally, I may want to try using a higher concentration of sera such as 1:100 instead of 1:500. I should also print plain e coli lysate as the negative control rather than induced e coli with PUC19. The spots should also be printed farther apart. Last time they were printed 350 um apart, but they should probably at least be printed 500 um apart.