1st+experiment+2-23-12

This will be the first experiment in which I apply sera onto the tumor cDNA expression library arrays. experiment actually performed on 2-29-12

SMC1fs, 4T1 tumor, naïve sera and secondary (AF647) were applied to array. Sera at 1:500 dilution. Secondary at 5 nM. Conditions: overnight, 23 C, Super G blocking buffer. Obtained some candidate spots for further screening.

Kathy mentioned that I may want to use a lower dilution (more antibody) for the tumor-immune sera than the anti-SMC1fs sera since the tumor reactivity may be low.

Samples 2 slides Mouse anti-SMC1fs antibody (just to show that my SMC1fs positive control cell lysate spots really are recognized by SMC1fs antibody) 2 slides tumor mouse (this sera would come from the mice which had the tumor from which this tumor cDNA library was constructed) 2 slides naive mouse 1 slides torino naive 1 slides torino no tumor 1 secondary only

Data is Stored here: C:\kurt\storage\CIM Research Folder\DR\2012\8-25-12\8-25-12_1402\Application of sera onto array\cell lysate 3-2-12 and here C:\kurt\storage\CIM Research Folder\DR\2012\3-2-12

Location of image of location of smc1fs and puc19 spots also location of positive controls L:\storage\CIM Research Folder\kwhittem\Records in CIM Folder\Categorical Records\Biodesign\Immunosignature to Antigen Bridge Project\Tumor cDNA Library\Application of sera onto array\cell lysate 3-2-12\smc1fs and puc19 locations

Powerpoint Summaries "C:\kurt\storage\CIM Research Folder\DR\2013\2-2-13\data_download\1112\Cell Lysate Array Results 3-6-12.ppt" "C:\kurt\storage\CIM Research Folder\DR\2013\2-2-13\data_download\1112\Cell Lysate Array Results 2 3-6-12.ppt" "C:\kurt\storage\CIM Research Folder\DR\2013\2-2-13\data_download\1112\Cell Lysate Array Results 4 3-6-12.ppt"

Note about analysis: choosing the top lysate spots is best performed by ranking the spots by the p-value from a t test comparing the sample against a control such as a naive sample. Choosing spots based on fold change or order of highest intensity does not work as well. e-mail to Kathy about analysis 3-7-12

Some Notes from Kathy about next steps 3-6-12 dot 1134

Note that 576 spots of 3072 (384*8) spots hava P value<0.05 when comparing tumor to normal. 567 of these decrease and 8 of these increase.

4-1-12 Note: After a presentation in the cancer meeting, people suggested that I reanalyze the data with median normalizations and secondary subtraction. I did this but the results were not very good (the SMC1fs spot was not in the top 10 or near the top 10 for the SMC1fs sera). I also tried looking at the data with normalization without the secondary subtraction, but this was also not good.

Some diagrams "C:\kurt\storage\CIM Research Folder\DR\2013\2-2-13\data_download\1112\Raw Intensity of tumor slides 2-23-12.svg" "C:\kurt\storage\CIM Research Folder\DR\2013\2-2-13\data_download\1112\Analysis of SMC1fs Spot.svg"

see false discovery analysis "C:\kurt\storage\CIM Research Folder\DR\2012\5-23-12\False discovery rate analysis of several experiments.docx"

I need to compare the differences between the feb experiment and the may experiment and pick a tumor spot to expand out onto a new array. "C:\kurt\storage\CIM Research Folder\DR\2012\5-24-12\Comparison of cDNA library array experiments.docx" SMC1fs, 4T1 tumor, naïve sera and secondary were applied to array. Sera at 1:500 dilution. Secondary at 5 nM. Conditions: overnight, 23 C, Super G blocking buffer. Obtained some candidate spots for further screening.