ProcartaPlex™ Human Akt Pathway Panel, phospho, 8plex

The panel is validated for use with cell lysates. For preparation of cell lysates, we recommend the use of either ProcartaPlex Cell Lysis Buffer (Cat. No. EPX-999999-900) or Cell Extraction Buffer (Cat. No. FNN0011) supplemented with 0.5 M EDTA and Halt Protease and Phosphatase Inhibitor Single–Use Cocktail (100X) (Cat. No. 78440 or 78442). These buffers are not included with the panel. Depending on your cell system, other buffers can be suitable as well.

The panel is provided in a ready-to-use format with individual vials of capture and detection reagents formulated at 1X concentration, requiring less pipetting and experimental setup.

ProcartaPlex preconfigured panels are extensively tested for analyte combinability, interference, and cross-reactivity to provide the highest level of validation and precision. All ProcartaPlex panels are supplied with the necessary reagents to perform the assay.

This kit is compatible with all Luminex instruments. If you use a xMAP INTELLIFLEX DR-SE instrument, we recommend using our ProcartaPlex Human Akt Pathway Dual Reporter Panel, 2x8plex (Cat.No. EPX080-97200-DR), which enables the simultaneous quantitation of total and phosphorylated proteins on the same bead in a single well.

Targets [bead region]: Akt[pS473] [47], CREB [PS133] [43], GSK-3 beta[pS9] [56], IGF-1R[pYpY1135/1136] [34], IRS-1[pS312] [37], mTOR[Ser2448] [26], PRAS40[pT246] [42], p70S6K[pTpS421/424] [27]

About ProcartaPlex Assays for the Luminex Platform

ProcartaPlex immunoassays are based on the principles of a sandwich ELISA, using two highly specific antibodies binding to different epitopes of one protein to quantitate all protein targets simultaneously using a Luminex instrument. ProcartaPlex multiplex assays require as little as 25 μL of plasma or serum or 50 μL of cell culture supernatant and just four hours to obtain analyzed results.

• More results per sample—measure multiple protein targets simultaneously in a single 25–50 μL sample
• Well-established Luminex technology—highly referenced multiplexing platform for protein detection and quantitation

ProcartaPlex assays utilize Luminex xMAP (multianalyte profiling) technology for the simultaneous detection and quantitation of up to 80 protein targets in a single 25–50 μL sample from plasma, serum, cell culture supernatant, cell lysate, and other bodily fluids.

The Luminex beads in the ProcartaPlex assay are internally dyed with precise proportions of red and infrared fluorophores to create spectrally unique signatures that can be identified by the Luminex xMAP detection systems (e.g., Luminex 200, FLEXMAP 3D, xMAP INTELLIFLEX, xMAP INTELLIFLEX DR-SE, and MAGPIX). Similar to a sandwich ELISA, the ProcartaPlex assay uses matched antibody pairs to identify the protein of interest. In a multiplexed assay, each spectrally unique bead is labeled with antibodies specific for a single target protein, and bound proteins are identified with biotinylated antibodies and streptavidin–R-phycoerythrin (RPE). The conjugation of protein-specific antibodies to a distinct bead allows for analysis of multiple targets in a single well.

In addition, the xMAP INTELLIFLEX DR-SE System offers dual-reporter capability, allowing the measurement of a second parameter of the analyte, and features side eject functionalities for added flexibility and automation. Together with ProcartaPlex signaling assays, the xMAP INTELLIFLEX DR-SE instrument enables the quantitation and multiplexing of phosphorylated and total proteins in the same well, on the same Luminex bead, and in the same assay run.

The most significant difference between a ProcartaPlex assay and ELISA is that the capture antibody in the ProcartaPlex assay is conjugated to a bead and not adsorbed to the microplate well, so the ProcartaPlex assay reagents are free-floating in the solution. For detection, the Luminex 200 instrument, for example, contains two lasers, one to distinguish the spectral signature of each bead and the second to quantify the amount of RPE fluorescence, which is proportional to the amount of protein present in the sample. ProcartaPlex multiplex assays can profile more target proteins using significantly less sample in the same time that it takes to perform a traditional sandwich ELISA.