In reconciling these observations particularly when the degree of phosphorylation of the epitopes is very low. In the present study, we described several easy, and inexpensive approaches that can be utilized to filter out the non-specific signal and improve tau signal specificity. Dynabeads, protein G, and the heat stable fraction all removed the non-specificity observed; however, we favor the use of secondary antibodies specific to native Igs or the use of anti-LC antibodies as these procedures interfered the least with our standard protocol and yielded good results. Finally, we stress the importance of using both negative and positive controls to ascertain the specificity of a given signal during Western blot analysis. Malignant gliomas are highly aggressive and uniformly lethal human brain cancers for which tumor recurrence following conventional therapies remains a major challenge for successful treatment. Immunotherapy is emerging as a promising therapeutic approach due to its potential to specifically seek-out and attack malignant cells, particularly the infiltrated cells often responsible for disease recurrence, while sparing cells of the normal brain parenchyma. For this reason, significant efforts are dedicated towards identifying targets amenable for immunotherapy of brain tumors. One attractive immunotherapy target is IL13Ra2, a 42-kDa monomeric high affinity IL-13 receptor distinct from the more ubiquitously expressed IL-13Ra1/IL-4Ra receptor complex. IL13Ra2 is expressed by a high percentage of gliomas, but not at significant levels on normal brain tissue, and in IL13Ra2expressing tumors has been identified on both stem-like malignant cells and their more differentiated counterparts. Targeting IL13Ra2 is currently the focus of ongoing clinical development for the treatment of brain tumors. In one such effort, our group has constructed an IL13 -zetakine CAR for targeting IL13Ra2. Expanded ex vivo, IL13-zetakine+ CTL retain MHC-independent IL13Ra2-specific anti-glioma cytolytic activity, maintain CAR-regulated Tc1 cytokine secretion and proliferation, and mediate regression of established human glioblastoma xenografts in vivo. These pre-clinical studies have culminated in a FDA-authorized feasibility/safety clinical trial of intracranial adoptive therapy with autologous IL13-zetakine + CD8 + CTL clones targeting recurrent/progressive malignant glioma. Because various combinations of cytokines have been reported to induce IL13Ra2 on a variety of cell types, we reasoned that using similar protocols to increase surface expression of IL13Ra2 on glioma cells would enhance therapeutic efficacy of multiple IL13Ra2-targeting treatment modalities including IL13-zetakine + CTLs. However, in the course of these studies we obtained divergent results with two IL13Ra2-directed antibodies: a goat polyclonal antibody from R&D Systems and a PE-conjugated mouse monoclonal antibody clone B-D13 from Cell Sciences.