Akt is a serine/threonine kinase that requires a functional phosphatidylinositol 3-kinase to be stimulated by insulin and other growth factors. When directed to membranes by the addition of a src myristoylation sequence, Akt becomes constitutively active. In the present study, a conditionally active version of Akt was constructed by fusing the Akt containing the myristoylation sequence to the hormone binding domain of a mutant murine estrogen receptor that selectively binds 4-hydroxytamoxifen. The chimeric protein was expressed in NIH3T3 cells and was shown to be stimulated by hormone treatment 17-fold after only a 20-min treatment. This hormone treatment also stimulated an approximate 3-fold increase in the phosphorylation of the chimeric protein and a shift in its migration on SDS gels. Activation of this conditionally active Akt resulted in the rapid stimulation of the 70-kDa S6 kinase. This conditionally active Akt was also found to rapidly stimulate in these cells the phosphorylation of properties of PHAS-I, a key protein in the regulation of protein synthesis. The conditionally active Akt, when expressed in 3T3-L1 adipocytes, was also stimulated, although its rate and extent of activation was less then in the NIH3T3 cells. Its stimulation was shown to be capable of inducing glucose uptake into adipocytes by stimulating translocation of the insulin-responsive glucose transporter GLUT4 to the plasma membrane.

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The IMAP Fluorescence Polarization technology is a homogeneous antibody-free method for analysis of kinases, phosphatases, and phosphodiesterases. Recent developments to the technology include an enhancement of the reagent system (the Progressive Binding System) that significantly expands the range of useable concentrations of ATP, choices of substrates, and assay configurations. With the new Progressive System, we are able to design multiplexed assays that allow the simultaneous determination of multiple kinase activities. In addition, coupled assays are now possible, allowing the assay of kinases through natural or artificial coupling through kinase cascades.

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Detection and quantification of proteins and their post-translational modifications are crucial to decipher functions of complex protein networks in cell biology and medicine. Capillary isoelectric focusing together with antibody-based detection can resolve and identify proteins and their isoforms with modest sample input. However, insufficient sensitivity prevents detection of proteins present at low concentrations and antibody cross-reactivity results in unspecific detection that cannot be distinguished from bona fide protein isoforms. By using DNA-conjugated antibodies enhanced signals can be obtained via rolling circle amplification (RCA). Both sensitivity and specificity can be greatly improved in assays dependent on target recognition by pairs of antibodies using in situ proximity ligation assays (PLA). Here we applied these DNA-assisted RCA techniques in capillary isoelectric focusing to resolve endogenous signaling transducers and isoforms along vascular endothelial growth factor (VEGF) signaling pathways at concentrations too low to be detected in standard assays. We also demonstrate background rejection and enhanced specificity when protein detection depended on binding by pairs of antibodies using in situ PLA, compared to assays where each antibody preparation was used on its own.Capillary electrophoresis (CE), coupled with fluorescence detection, liquid chromatography or mass spectrometry, has many applications in separating and detecting biomolecules such as DNA 1 , metabolites 2 , peptides 3 and proteins 4 , since it offers high resolution and uses small amounts of samples. In particular, capillary isoelectric focusing (IEF) has proven useful for resolving protein isoforms such as phosphorylated variants 5 , since they typically have different isoelectric points (pI), allowing their separation in an ampholyte gradient gel.The NanoPro 1000 system from ProteinSimple is a recently developed automated capillary IEF system where proteins are first separated and then immobilized in capillaries, followed by antibody probing in analogy to standard immunoblotting for protein identification and quantification 6 . However, the system differs from immunoblotting in two important respects 7 : First, in conventional immunoblotting proteins are separated according to molecular weight, while separation by IEF depends on the charge of the investigated protein species. Secondly, in immunoblotting, proteins are denatured by treatment with an ionic detergent while proteins separated by capillary IEF remain in a native state, which may influence the ability of antibodies to recognize the proteins. The capillary IEF method permits the use of limited tissue samples, as the technique efficiently resolves and quantifies proteins and their isoforms in submicroliter samples. Native proteins extracted from lysates of cells or tissues are separated according to charge, resolving isoforms of the proteins, whereupon the proteins are immobilized on