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