Cdc42 G15A Agarose Beads (Active Cdc42-GEF) (ab211185)
Overview
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Product name
Cdc42 G15A Agarose Beads (Active Cdc42-GEF)
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Product overview
Background: Small GTP-binding proteins (or GTPases) are a family of proteins that serve as molecular regulators in signaling transduction pathways. Cdc42, 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, Cdc42 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. Cdc42 G15A Agarose beads to selectively isolate and pull-down the active form of Cdc42-GEFs from purified samples or endogenous lysates. Subsequently, the precipitated Cdc42-GEF is detected by western blot analysis using an anti-Cdc42-GEF antibody.Use: Our Cdc42 G15A Agarose Beads are designed to pull down only the active form of Cdc42 GEF.
Description: Cdc42 G15A Agarose beads, in color, are easy to visualize, minimizing potential loss during washes and aspirations of active Cdc42-GEF pulldown.
Purity and Activity: Purity >90% by SDS-PAGE and Coomassie blue staining. Specifically interacts and precipitaes active
Cdc42-GEF from cell lysate.Concentration: 800 μL of 50% Agarose slurry, 1 mg/mL Cdc42 G15A in 1X PBS, 50% Glycerol
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Notes
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
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Storage instructions
Store at -20°C. Please refer to protocols. -
Components 800 µg Cdc42 G15A Agarose Beads (Active Cdc42-GEF) 1 x 800µg -
Research areas
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Function
Plasma membrane-associated small GTPase which cycles between an active GTP-bound and an inactive GDP-bound state. In active state binds to a variety of effector proteins to regulate cellular responses. Involved in epithelial cell polarization processes. Causes the formation of thin, actin-rich surface projections called filopodia. -
Sequence similarities
Belongs to the small GTPase superfamily. Rho family. CDC42 subfamily. -
Post-translational
modificationsAMPylation at Tyr-32 and Thr-35 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. -
Cellular localization
Cell membrane. - Information by UniProt
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Alternative names
- CDC42
- CDC42_HUMAN
- CDC42Hs
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