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Cat. No.: R1RAG-1 (for 1ml)

Description

Raf1-RBD Agarose, also known as Raf1-RBD agarose gel, is an agarose gel prepared by coupling the GST-tagged recombinant human Raf1 RBD domain protein expressed in E. coli with glutathione agarose (GST agarose). This product is designed for the active Ras pull-down assay, allowing for the binding, precipitation, or pull-down of active state Ras proteins (including NRas, HRas, and KRas). It can be used to detect the ratio of activated Ras proteins to inactive states in vitro, as well as for quantitative detection of active Ras proteins in tissue or cell lysates.

Raf1 is a downstream effector protein that interacts with the Ras subfamily, including NRas, HRas, and KRas. It plays a crucial role in cell proliferation, differentiation, apoptosis, survival, and oncogenic transformation. Raf1 consists of 648 amino acids with a theoretical molecular weight of 73 kDa and contains four domains: the Active Ras-binding domain (RBD), the Cysteine-rich domain (CRD), an inhibitory phosphorylation site involved in Raf1 activation, and a kinase domain. The RBD of Raf1 specifically recognizes and binds to active Ras proteins but does not bind to inactive Ras proteins, making Raf1-RBD recombinant protein useful for detecting active Ras proteins. It can also serve as an inhibitor of Ras proteins both in vitro and in vivo.

Small GTPases, also known as Small G-proteins or the Ras superfamily, act as molecular switches that regulate various processes in eukaryotic cells, including signal transduction, cell proliferation, cytoskeletal remodeling, and intracellular membrane transport. Small GTPases cycle between active and inactive states through the binding and hydrolysis of GTP. In response to external signals, guanine nucleotide-exchange factors (GEFs) assist small GTPases in replacing bound GDP with GTP, activating the GTPase. The active small GTPase interacts with downstream effector proteins to elicit cellular responses. GTPase-activating proteins (GAPs) catalyze the hydrolysis of GTP to GDP, releasing free phosphate (Pi) and returning the small GTPase to its inactive state. Guanine nucleotide dissociation inhibitors (GDIs) prevent the release of GDP until GEFs are stimulated again, initiating a new cycle.

More than 150 family members of small GTPases have been identified in humans, and conserved homologs have also been found in fruit flies (Drosophila), Caenorhabditis elegans, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Dictyostelium, and plants. Small GTPases are classified into five family branches based on their structure and function: the Ras family, Rho family, Ran family, Rab family, and Arf family. The Ras family itself is further divided into six subfamilies: the Ras subfamily, Ral subfamily, Rit subfamily, Rap subfamily, Rheb subfamily, and Rad subfamily.

The Ras family (Ras homologous GTPases) is a key factor in regulating actin remodeling, playing an important role in cellular processes such as maintaining the integrity of the actin cytoskeleton, cell proliferation, cell differentiation, cell adhesion, apoptosis, and cell migration. Currently, 16 members have been discovered, classified into six subfamilies (see table below), with NRas, HRas, and KRas being the most extensively studied.

Raf1-RBD Agarose Storage Solution: 50 mM HEPES (pH 7.4), 150 mM NaCl, 5 mM MgCl₂, 0.5% Triton X-100, 1 mM DTT, 10% glycerol.

Lysis Buffer: 50 mM Tris (pH 7.4), 1% Triton X-100, 0.5% Sodium Deoxycholate, 0.1% SDS, 500 mM NaCl, 10 mM MgCl₂, 10 μg/ml Leupeptin, 10 μg/ml Aprotinin, 1 mM PMSF.

Wash Buffer: 50 mM Tris (pH 7.4), 40 mM NaCl, 30 mM MgCl₂.

This product is packaged in a volume of 1 ml, with 50% of that volume consisting of settled gel, which combines approximately 0.5 mg of GST-Raf1-RBD recombinant protein. Based on using 20-50 μl of Raf1-RBD Agarose suspension per pull-down reaction, this product can be used for 20-50 reactions per 1 ml.

Storage

Store at -80ºC, valid for one year. Store at 4ºC, valid for one month.

Precautions

• Upon arrival, store at -20ºC or -80ºC. Freeze-thaw cycles are effective for up to two times; avoid repeated freeze-thaw. For the first use, aliquot as needed.
• This product is for scientific research use by professionals only and should not be used for clinical diagnosis or treatment, nor for food or pharmaceuticals, and should not be stored in a regular residence.
• For your safety and health, please wear lab coats and disposable gloves during operation.

Only for research and not intended for treatment of humans or animals

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