Product nameRalGDS RBD Agarose Beads
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.
Use: 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.
Storage instructionsStore at -20°C. Please refer to protocols.
RelevanceStimulates the dissociation of GDP from the Ras related RalA and RalB GTPases which allows GTP binding and activation of the GTPases. Interacts and acts as an effector molecule for R Ras, H Ras, K Ras, and Rap (from SwissProt).
Cellular localizationCytoplasmic vesicle
- ral guanine nucleotide dissociation stimulator
To our knowledge, customised protocols are not required for this product. Please try the standard protocols listed below and let us know how you get on.
ab211179 has not yet been referenced specifically in any publications.