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

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"