Tumor+vs+normal+on+Single+Clones+9-21-12

Tumor vs normal on Single Clones 9-21-12

actual experiment on 9-29-12

applied tumor and naïve sera to tumor library cell lysate arrays at 3 different dilutions (1:1000, 1:500, and 1:500). 1 nM secondary used. This experiment revealed that the tumor sera had higher intensity for the spots than the naïve sera at higher dilutions as I predicted from the previous experiment. Note that the Tecan had a “low pressure” error during this experiment, and we believe that the compressor failed for reasons unrelated to this particular experiment. The compressor just needs to be maintained.

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 printed onto the array as a negative control. The arrays will be probed with naive sera and tumor sera at different dilutions (1:1000, 1:500, and 1:100 sera).

planning started on 9-21-12 John and I think that we may repeat the previous array experiment (Tumor vs normal on Single Clones 9-12-12). The slides would be printed the same way. However, instead of just using the higher than normal concentration used here (1:100 used here as compared with 1:500), I will have 3 for 1:1000, 3 for 1:500, and 3 for 1:100 for a total of 9 slides. I will also make sure that the chambers are dried before I use them. I can observe if there is the same decrease in intensity with consecutively printed slides.

sera was applied to array on 9-29-12 and went overnight. Secondary was added on 9-30-12.

The Tecan had some type of problem related to low pressure. Zbig and Bart think that the compressor is bad. The error message can be found here "S:\Research\Cancer_Eradication\Users\kwhittem\DR\2012\9-30-12\tecan error messages 9-30-12"

The array images can be found here "S:\Research\Cancer_Eradication\Users\kwhittem\DR\2012\9-30-12\nv-tmr-lst-9-30-12"

I can use the same gps file from the last experiment to start the alignment.

Analysis can be found here "C:\kurt\storage\CIM Research Folder\DR\2012\10-2-12\analysis 10-1-12.xlsx" "C:\kurt\storage\CIM Research Folder\DR\2012\10-2-12\analysis of slide means.xlsx"

The 1:1000 slides were scanned again at a higher laser power since they had a lower intensity and were reanalyzed. Note that the previous scans had the autogain set which I did not realize. These new scans for the 1:1000 slides have a set gain. "C:\kurt\storage\CIM Research Folder\DR\2012\10-2-12\analysis 10-2-12.xlsx"

Summary here "C:\kurt\storage\CIM Research Folder\DR\2012\10-2-12\Summary Tumor Library Lysate 10-2-12.docx"

Summary for search indexing 10-2-12

Was there an improvement with reduction to single clones? analysis here "C:\kurt\storage\CIM Research Folder\DR\2012\10-12-12\array data\analysis of improvement to single clones 10-12-12.xlsx" Hard to say that there was an improvement, but there was some consistency depending on where the p value and fold change thresholds are set. "C:\kurt\storage\CIM Research Folder\DR\2012\10-12-12\array data\single clone vs pooled volcano 10-12-12.svg" "C:\kurt\storage\CIM Research Folder\DR\2012\10-12-12\array data\single clone vs pooled volcano 10-12-12.pdf"