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.