Tumor antibody purification with random peptides 8-3-12

2015-01-13

azim58 - Tumor antibody purification with random peptides 8-3-12

I made several modifications to the protocol I followed when I previously
tried to purify tumor antibodies.

used tentagel beads with 4X peptide density
used 1 mL sera (instead of 10 uL in 1000 uL buffer)
performed purification in 1.5 mL tube rather than column
incubated overnight at 4 C
cleared the sera before purification by incubating 10 min at 1000Xg

at RT

tumor sera used: tumor pooled from 6-18-12
needed extra sera as well
tumor sera 6-18-10 KW thawed 8-3-12 0.02% sodium azide

Item: Unbound tumor sera 8-4-12 KW
Item: Elution Fractions 1-5 8-4-12 KW
Item: Ab Purification 8-3-12 KW

Identity of peptides associated with this experiment can be found here:
Synthesis of Random Peptides for Antibody Purification 5-17-12

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8-7-12
A silverstain and a western blot was performed to check for the presence
of antibodies in the elution fractions.

Item: secondary Goat anti-mouse IgG H+L AF647 8-7-12 (1 to 10,000
dilution of 2 mg/mL solution)

At first, 1:1,000 goat anti-mouse IgG H+L was applied to the
nitrocellulose membrane for 1 hr at 37 C. I couldn't really see the
antibody band in my elution fractions after this. The results are here.
"L:\storage\CIM Research Folder\DR\2012\8-7-12\ab purification
western\western 8-7-12"
Then I incubated the membrane with 1:10,000 goat anti-mouse IgG H+L for 1
hr at 37 C, and I was able to detect the antibody. The results are here.
"C:\kurt\storage\CIM Research Folder\DR\2012\8-7-12\ab purification
western\western 2 8-7-12"

Silverstain results can be found here:
"L:\storage\CIM Research Folder\DR\2012\8-7-12\ab purification
western\silverstain of fractions 8-6-12"

A summary of the results can be found here:
"L:\storage\CIM Research Folder\DR\2012\8-7-12\ab purification
western\summary of ab detection.svg"
and here
"C:\kurt\storage\CIM Research Folder\DR\2012\8-7-12\ab purification
western\summary of ab detection.pdf"