Slides were then incubated for 32?minutes with anti-C2Cat diluted 1/300. in cells and tissues. Similar structure-based rational design strategies can be broadly applied to obtain active-state specific antibodies for other signal transduction molecules. The Protein kinase C (PKC) family of serine-threonine kinases is composed of at least ten isoenzymes, that play significant regulatory roles in key pathways in cancer such as, cell motility, survival, cell cycle and differentiation. Recent studies have exhibited that mutations in PKC usually lead to a loss of function suggesting a tumor suppressor role for PKCs. In some tumors, such as breast cancer, PKC mutations are rare1 and there are difficulties to establish the cause-effect JDTic of the PKC family of proteins. This may be due to complex biological activities involving the different isoenzymes coupled with difficulties in measuring expression and activation status of each isoenzyme2. Furthermore, there is generally a poor correlation between mRNA levels and protein expression levels, due to antibody specificity or data for mRNA levels generated from whole tissues instead of microdissected tumor cells. Since phosphorylation is usually a step for PKC protein folding (maturation) more JDTic than a marker of protein activation as known for Erk, Akt or JNK, we currently can only detect PKC isoenzyme activation status or substrate phosphorylation in cell cultures but not in tumor tissue2. Difficulties to determine PKC expression and activation status are partly due to the fact that this PKC family is composed of at least ten different isoenzymes divided into three sub-families according to their activation requirements. Classical (cPKCs): , , and are calcium dependent and activated by phosphatidyl serine and diacylglycerol , novel (nPKCs): are calcium impartial, but depend on the same lipids as cPKCs for their activation and atypical (aPKCs): and are calcium independent and activated JDTic by different lipids such as ceramide3. PKC activation requires translocation of the enzyme to membranes4 where it modulates signaling cascades through phosphorylation of serine and threonine amino acid residues of a large selection of proteins. Most antibodies that are thought to detect active kinases are directed against phosphorylation sites considered essential for kinase activation such as the activation loop5. These antibodies have been used in a variety of assays to determine cancer prognosis with limited success since tissue processing conditions may lead to loss of phosphate groups and false unfavorable signals6. Further, in the case of cPKCs, phosphorylation does not correlate with activation7,8,9. PKCs undergo three phosphorylations at specific sites in the catalytic domain name activation loop, turn motif, and hydrophobic motif. These phosphorylations confer stability, catalytic competence and proper subcellular localization of active kinase9. In cPKCs, these phosphorylations are present in inactive says and are a part of proper kinase folding. Complete activation of cPKCs requires binding to calcium and phospholipids (diacylglycerol and phosphatidyl serine)7,8,9. Upon activation, PKCs undergo conformational changes that prevent intramolecular interactions and expose regions involved with binding to substrates and scaffold proteins10,11. A classic example is the intramolecular conversation between the pseudo-substrate Vax2 (PS) site in the N-terminus of cPKCs and the catalytic domain name. Upon activation PKCs bind isoenzyme specific receptors for activated C kinases or RACKS. In an inactive enzyme the RACK binding site is also involved in an intramolecular conversation with a region named RACK, that is similar to a site in its corresponding RACK11. Both of these intramolecular interactions impair substrate and scaffold protein binding to the kinase, and are interrupted upon lipid binding and PKC activation11,12. Recently, (2015) the reinterpretation of a previously reported crystal structure of PKCII combined with docking of the different domains in the kinase, proposed an alternative structure for inactive PKC with the C2 domain name interacting with the carboxy-terminus (V5) and the catalytic domain name of the kinase further contributing to the maintenance of the kinase in an inactive conformation10. These findings shed light to the idea that it is possible to explore PKCs structure for epitopes that become uncovered only in an active kinase. A peptide derived from an conversation region between the C2 and catalytic domain name of cPKC in an inactive kinase was chosen and.