1st experiment 2-23-12

2014-08-29

azim58 - 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.

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