Tumor vs normal on Single Clones 8-27-12

2015-01-13

azim58 - 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.

azim58wiki: