Description
Key facts
Form:LiquidSee storage information,
Storage buffer:Constituents: 50% Glycerol (glycerin, glycerine)
Product details:
Background:
Small GTP-binding proteins (or GTPases) are a family of proteins that serve as molecular regulators in signalling transduction pathways. Ras, a 21 kDa protein, regulating a variety of biological response pathways that include cell growth, cell transformation and tumour invasion. Like other small GTPases, Ras regulates molecular events by cycling between an inactive GDP-bound form and an active GTP-bound form. In its active (GTP-bound) state, Ras binds specifically to the Ras-binding domain (RBD) of Raf1 to control downstream signalling cascades.
Our Raf-1 RBD Agarose Beads are designed to pull down only the active form of Ras.
Description:
Our Raf RBD Agarose beads are colored for easy visualization, minimizing potential loss during washes and aspirations during Ras-GTP pulldown.
Activity:
Product specifically interacts and precipitates GTP-bound Ras from cell lysate.
Concentration:
800 μL of 50% Agarose slurry, 400 μg Raf1-RBD in 1X PBS, 50% Glycerol
Protocol for the pull down assay:
1. Aliquot 0.5 – 1 mL of cell lysate to a microcentrifuge tube.
2. Adjust the volume of each sample to 1 mL with 1X lysis buffer.
3. Thoroughly resuspend the agarose bead slurry by vortexing or titrating.
4. Quickly add 40 μL of resuspended bead slurry to each tube.
5. Incubate the tubes at 4°C for 1 hour with gentle agitation.
6. Pellet the beads by centrifugation for 10 seconds at 14,000 x g.
7. Aspirate and discard the supernatant, making sure not to disturb/remove the bead pellet.
8. Wash the bead 3 times with 0.5 mL of 1X lysis buffer, centrifuging and aspirating each time.
9. After the last wash, pellet the beads and carefully remove all the supernatant.
10. Resuspend the bead pellet in 40 μL of 2X reducing SDS-PAGE sample buffer.
12. Boil each sample for 5 minutes.
13. Centrifuge each sample for 10 seconds at 14,000 x g.
For best results with these beads, it is important to first determine the amount of cell lysate that is detectable on the blot before performing the pull down. We recommend running a lysate titration on a Western blot to determine the concentration that gives a good signal. For the GTPase assay, you will then want to add 100-fold that amount. For example, if you run 5, 10 and 20ug of lysate on a Western blot and 10ug gives a good signal, you will use 10ug x 100 = 1mg of lysate per pull down.
The activity level of the small GTPase in the sample will determine how much gets pulled down. The beads are designed to only pull down small GTPase in the GTP-bound (active) form. If the majority of the GTPase in the sample is in the GDP-bound form (inactive), it will not get pulled down, regardless of the amount of lysate loaded. The lysate can be preloaded with GTPγS and used as a positive control.
Sequence alignment of a specific small GTPase indicates that there is at most one or two amino acid variation between various species. Therefore, our beads may be used across many species.
Properties and Storage Information:
Shipped at conditions-Blue Ice, Appropriate short-term storage conditions--20°C, Appropriate long-term storage conditions--20°C
Supplementary Information:
This supplementary information is collated from multiple sources and compiled automatically.
Raf1 also known as c-Raf or Raf-1 is a serine/threonine-protein kinase that plays an important role in cell division differentiation and survival. Its molecular weight is approximately 74 kDa. Raf1 is part of the MAPK/ERK signaling pathway and is ubiquitously expressed in many tissues including the brain heart and liver. As a component of the signaling cascade its activation translates extracellular signals into intracellular responses.
Biological function summary
Raf1 regulates important cellular processes by activating downstream kinases in response to external stimuli. Raf1 forms a complex with other proteins such as Ras facilitating its role as an essential component of the MAPK/ERK pathway. Upon activation by Ras Raf1 phosphorylates and activates MEK1 and MEK2 which in turn activate the ERK1 and ERK2. This signaling axis is involved in controlling gene expression and cellular proliferation.
Pathways
Raf1 is integrally involved in the MAPK/ERK pathway which is critical for transducing signals from growth factors and mitogens. It relates closely with proteins such as Ras MEK and ERK in this pathway. The pathway is important for regulating cellular responses to various stimuli and is particularly involved in processes such as cell cycle control and apoptosis.
Raf1 has associations with certain types of cancer and Noonan syndrome. In various cancers mutations or dysregulation of Raf1 or related components such as Ras can lead to uncontrolled cellular proliferation. In Noonan syndrome Raf1 mutations result in anomalies in the Ras/MAPK pathway which can impact normal development. Raf1’s interactions in these pathways indicate its relevance as a potential target for therapeutic intervention.
