Tumor vs normal on Single Clones 9-12-12

2015-01-13

azim58 - Tumor vs normal on Single Clones 9-12-12

9-12-12

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 printed onto the array as a
negative control. The arrays will be probed with naive sera and tumor
sera.

I am making several modifications for this print run as compared to the
last print run. I am only printing each type of spot 2 times in each half
chamber so that antibodies don't become very spread out among the spots.
The spots will also be far apart on the array instead of right next to
eachother. This is going to be accomplished by printing with lysate from
two 384 well plates that have the same samples in the reverse order. I
will use a higher concentration of sera (1:100 instead of 1:500); the
secondary will remain at 1 nM. E coli lysate will be printed as the
negative control instead of PUC19. The spots will be more than 500 um
apart (about 1-1.5 mm apart).

For print run details see Tumor Lysate Nitrocellulose Print 9-12-12

Experiment started on 9-14-12
an overnight 16 hour incubation was performed for the primary sera
on 9-15-12 the secondary was added and the slides were scanned. Note that
the slides had to be scanned at a low laser power (1% laser power 20%
gain) otherwise some of the spots were at the highest intensity.

location of scanned files
(the slides were scanned on 9-15-12 even though the date reads 9-16-12)
S:\Research\Cancer_Eradication\Users\kwhittem\kwhittem\Raw Data Often
Originally on Research Drive\2012\9-16-12 nv tmr scan

Summary here
"C:\kurt\storage\CIM Research Folder\DR\2012\9-17-12\Summary of 9-17-12
Experiment.docx"
Excel file here
"S:\Research\Cancer_Eradication\Users\kwhittem\kwhittem\Raw Data Often
Originally on Research Drive\2012\9-16-12 nv tmr scan\results\results
9-17-12.xlsx"

I expected that increasing the concentration of the sera (from the
original 1:500 to 1:100) would increase the intensity of the tumor spots
compared to naïve. However, the opposite occurred. Perhaps there is more
antibody in the naïve sera overall and increasing the concentration
resulted in more binding. Diluting the sera may result in more tumor
binding than naïve binding. Alternatively, measuring the amount of
antibody in both types of sera and diluting so that they are the same may
be the best approach.

Note that one other slight possibility is that there was some residual
antibody or sample leftover from the previous Tecan use. This is probably
unlikely since the chambers were washed after use. However, I used the
chambers before they had been completely dried out. Therefore, there was
still some liquid in the microfluidic tubes in the chamber, and this
liquid may have contained some residual material. The naive would have
had consistently higher intensity than the normal because the samples
were added in an alternating top and bottom manner which is the same way
that others add their samples to the Tecan. Therefore, if the previous
Tecan user had a sample with a lot of strongly binding antibody, they
could have added this sample in the same order that I added my naive
sample. So there is a small possibility that simply repeating the
experiment with dried chambers could yield better results.

Note that one of the chambers yielded tumor intensities that were higher
than naive while all of the other chambers yielded naive intensities that
were higher than tumor.

I can check for evidence of contamination by looking at the previous run
on the Tecan by Zbig.
"C:\kurt\storage\CIM Research Folder\DR\2012\9-19-12\array\Layout of
Zbig's array run 9-14-12.pdf"
Images of slides found here
S:\Administration\PeptideArrayCore\2012 sample
run\DTRA-2\Diseases-5-8_run\09142012

Analysis for contamination found here
C:\kurt\storage\CIM Research Folder\DR\2012\9-20-12\Array

see:
how to measure amount of antibody in a sample

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