Product nameRhoA G17A Agarose Beads (Active Rho-GEF)
Background: Small GTP-binding proteins (or GTPases) are a family of proteins that serve as molecular regulators in signalling transduction pathways.Rho, a 21 kDa protein, belongs to the family of Rho GTPases regulating a variety of biological response pathways that include cell motility, cell division, gene transcription, and cell transformation. Like other small GTPases, Rho influences molecular events by cycling between an inactive GDP-bound form and an active GTP-bound form. Cycling between the GDP-bound and GTP-bound state is regulated primarily by two distinct families of proteins: guanine nucleotide exchange factors (GEFs) activate Rho proteins by catalyzing the exchange of GDP for GTP, the GTPase activating proteins or GAPs negatively regulate GTPase function by stimulating GTP hydrolysis.
Similar to Ras mutants, constitutively active or dominant negative Rho GTPase mutants have been used to bind to Rho-GAP and effectors or to Rho-GEFs, respectively. A nucleotide-free GTPase has also been shown to form a high affinity binary complex with Rho-GEFs. RhoA G17A Agarose beads selectively isolate and pull-down the active form of Rho-GEFs from purified samples or endogenous lysates. Subsequently, the precipitated Rho-GEF is detected by western blot analysis using an anti-Rho-GEF antibody.
Use: Our RhoA G17A Agarose beads are designed to pull down only the active form of RhoA-GEF.
Description: Our RhoA G17A beads are colored for easy visualization, minimizing potential loss during washes and aspirations during RhoA GEF pulldown.
Purity and Activity: Purity >90% by SDS-PAGE and Coomassie blue staining. Specifically interacts and precipitaes active Rho-GEF from cell lysate.
Concentration: 1 mL of 50% Agarose slurry, 0.4 mg/mL RhoA G17A 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.
Components 400 µg RhoA G17A Agarose Beads (Active Rho-GEF) 1 x 400µg
FunctionRegulates a signal transduction pathway linking plasma membrane receptors to the assembly of focal adhesions and actin stress fibers. Serves as a target for the yopT cysteine peptidase from Yersinia pestis, vector of the plague, and Yersinia pseudotuberculosis, which causes gastrointestinal disorders. May be an activator of PLCE1. Activated by ARHGEF2, which promotes the exchange of GDP for GTP.
Sequence similaritiesBelongs to the small GTPase superfamily. Rho family.
DomainThe basic-rich region is essential for yopT recognition and cleavage.
modificationsSubstrate for botulinum ADP-ribosyltransferase.
Cleaved by yopT protease when the cell is infected by some Yersinia pathogens. This removes the lipid attachment, and leads to its displacement from plasma membrane and to subsequent cytoskeleton cleavage.
AMPylation at Tyr-34 and Thr-37 are mediated by bacterial enzymes in case of infection by H.somnus and V.parahaemolyticus, respectively. AMPylation occurs in the effector region and leads to inactivation of the GTPase activity by preventing the interaction with downstream effectors, thereby inhibiting actin assembly in infected cells. It is unclear whether some human enzyme mediates AMPylation; FICD has such ability in vitro but additional experiments remain to be done to confirm results in vivo.
Ubiquitinated by the BCR(BACURD1) and BCR(BACURD2) E3 ubiquitin ligase complexes, leading to its degradation by the proteasome, thereby regulating the actin cytoskeleton and cell migration.
Cellular localizationCell membrane. Cytoplasm > cytoskeleton.
- Information by UniProt
- Aplysia ras related homolog 12
ab211183 has not yet been referenced specifically in any publications.