Tumor Antibody Dot Blot 8-12-12

2015-01-13

azim58 - Tumor Antibody Dot Blot 8-12-12

Tumor Antibody Dot Blot 8-12-12

I will determine whether my purified antibody sera binds to the 6 random
peptides found here: Synthesis of Random Peptides for Antibody
Purification 5-17-12
I will refer to the dot blot protcol

I will spot the 6 random peptides as well as the SIINFEKL peptide as a
negative control.
I will dilute the peptides in water. If they are not soluble, I will use
40% acetonitrile.
Two membranes will be spotted. One will probed with tumor sera, and one
will be probed with naive sera.
What concentration or amount of peptide or protein should be spotted in a
dot blot?

A quick search did not reveal this information.
I will use typical protein western blot ladders as a guide. . <-

actually companies don't seem to state the amount of protein.

I will try to blot about 1000 ng of protein since I could detect this

fairly easily in a previous Western in which I was trying to detect the
amount of IgG present in some sera.

Item: 6 peptides diluted in 40% acetonitrile 8-12-12
see Handling Peptides

Initial unorganized results can be found here
"L:\storage\CIM Research Folder\DR\2012\8-13-12\tumor ran peptide db
8-13-12"
and here
L:\storage\CIM Research Folder\DR\2012\8-15-12\dot blot

Analysis of Results
"L:\storage\CIM Research Folder\DR\2012\8-16-12\dot blot\Analysis of Dot
Blot 8-16-12.docx"
Summary of Results
"L:\storage\CIM Research Folder\DR\2012\8-16-12\dot blot\dot blot summary
8-16-12.svg"
Text for indexing 8-16-12 dot 1146
Both the naive and tumor samples had about the same intensity for some of
the 6 random peptides. Both types of sera
displayed high intensities for the NRVWW, YNPIW, and VNGEY peptides. The
VNGEY peptide was the only peptide for which
the tumor sera had higher intensity than the naive sera. Both sera types
had a low intensity for the negative control SIINFEKL
peptide. One thing to keep in mind is that the tumor sera was essentially
diluted 1 million fold (1000 fold dilution during the
antibody purification since the peptide was eluted in a 1 mL fraction and
1000 fold for the dot blot assay). The naive sera was only
diluted 1000 fold for the dot blot assay. Therefore, the two sera types
are not directly comparable. If I want a better comparison,
I could redo the dot blot assay by diluting the naive sera 1 million fold
or even purify the naive sera using the same random peptide
tentagel bead method, and then perform the dot blot.