Tumor vs normal on Single Clones 8-27-12
2015-01-13azim58 - 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.
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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.