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. Rap, a 24 kDa protein of the Ras superfamily, regulates a variety of biological response pathways that include cell adhesion, proliferation, differentiation, and apoptosis. The Ras-like proteins Rap1 and Rap2 share 60% identity. Like other small GTPases, Rap regulates molecular events by cycling between an inactive GDP-bound form and an active GTP-bound form. In their active (GTP-bound) state, Rap1 and Rap2 bind specifically to the Rap-binding domain (RBD) of RalGDS to control downstream signaling cascades.
Our RalGDS RBD Agarose beads are designed to pull down only the active form of Rap.
Description:
Our RalGDS RBD Agarose beads are colored for easy visualization, minimizing potential loss during washes and aspirations during Rap-GTP pulldown.
Activity:
Product specifically interacts and precipitates GTP-bound Rap from cell lysate.
Concentration:
800 μL of 50% Agarose slurry, 400 μg murine RalGDS-RBD (amino acid 726-823) 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.
RALGDS also known as Ral guanine nucleotide dissociation stimulator functions as a guanine nucleotide exchange factor (GEF) for Ral GTPases. With a molecular weight of approximately 115 kDa RALGDS resides primarily in the cytoplasm and various tissue types including the heart brain and kidneys. The alternative names for RALGDS include RGL1 and RalGEF. Its role entails facilitating the conversion of Ral proteins from an inactive GDP-bound state to an active GTP-bound state. This activation is critical for transmitting signals from cell surface receptors to various intracellular pathways.
Biological function summary
RALGDS interacts with Ras-related GTPases where it forms complexes that modulate cytoskeletal dynamics and vesicular trafficking. It does not solely operate within these complexes but its interaction is essential to effectively mediate downstream signaling events. RALGDS can influence cellular processes like proliferation migration and cytoskeleton organization through these mechanisms.
Pathways
It participates in the Ras signaling and Ral GTPase signaling pathways. These pathways contribute to the regulation of cell division and cell movement which are vital for normal cellular function and development. In this context RALGDS connects with other Ras-related proteins like Raf and PI3K which further propagate signaling cascades essential for cellular growth and differentiation.
RALGDS associates with cancer and neurological disorders. It plays a role in the progression of several cancers including lung and prostate cancer often through its interaction with Ras and RalA/B proteins. Additionally abnormalities in RALGDS signaling have potential links to Alzheimer’s disease as its signaling can impact pathways related to neuronal function. Understanding these interactions helps researchers target therapeutic interventions for these diseases.
