MILLIPLEX^® Ras-Raf Oncoprotein Magnetic Bead Panel 6-Plex - Cell Signaling Multiplex Assay

Ras proteins are GTPases encoded by three genes: HRAS, KRAS, and NRAS. The protein products of these genes exhibit a high degree of sequence identity and are regulated by a conserved mechanism: exchange of GDP for GTP switches the Ras protein into an active conformation. In its GTP-bound state, Ras has high affinity for its downstream effectors, including the Raf family of protein kinases (ARAF, BRAF, and CRAF/RAF1). Raf proteins, in turn, signal through MEK to activate the MAPK pathway. In healthy cells, Ras activation is a transient response to extracellular growth stimuli and is restricted by the rapid conversion of Ras-GTP back to Ras-GDP. However, the Ras-Raf-MEK pathway is often aberrantly activated in cancer. Mutations in the RAS genes are amongst the most frequently encountered oncogenic events, occurring in ~25% of human cancers, with mutations altering the 12th amino acid (glycine) being particularly prevalent. These driver mutations maintain Ras in a constitutively active, GTP-bound state. Additionally, certain cancers feature KRAS gene amplification, although the effects of gene amplification on protein expression are not yet well-characterized.The MILLIPLEX^® MAP 6-plex Ras-Raf Oncoprotein Magnetic Bead Kit (Cat. No. 48-684MAG) is a premixed kit to be used for the simultaneous quantification of the following 6 protein analytes:pBRAF (Ser446)pCRAF (Ser338)pMEK1 (Ser217/Ser221)Ras (total)Mutant Ras protein, RasG12D*Mutant Ras protein, RasG12V**Detects H-Ras, N-Ras, and K-Ras protein

Cross-reactivity between the antibodies and any of the other analytes in this panel is non-detectable or negligible.

Intracellular Bead-Based Multiplex Assays using the Luminex technology enable the simultaneous relative quantitation of multiple phosphorylation and total pathway proteins in tissue and cell lysate samples. Compare Multiplexing results to those of Western blotting.An overnight (4°C) incubation is recommended for best results.This kit must be run using Assay Buffer 2 (provided).This assay requires 25 μL diluted cell lysate per well.The suggested working range of protein concentration for the assay is 1 to 25 μg of total protein/well (25 μL/well at 40 to 1,000 μg/mL).Analytes available:BRAF (Ser446);CRAF (Ser338);MEK1 (Ser217/Ser221);Ras (total);RasG12D;RasG12V

Configuration: Premixed

96-well plate

Recommended storage for kit components is 2 - 8°C.

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MILLIPLEX is a registered trademark of Merck KGaA, Darmstadt, Germany

xMAP is a registered trademark of Luminex Corp

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