Product nameRac1 G15A Agarose Beads (Active Rac-GEF)
Background: Small GTP-binding proteins (or GTPases) are a family of proteins that serve as molecular regulators in signaling transduction pathways. Rac, 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, Rac 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. Rac1 G15A Agarose beads selectively isolate and pull-down the active form of Rac-GEFs from purified samples or endogenous lysates. Subsequently, the precipitated Rac-GEF is detected by western blot analysis using an anti-Rac-GEF antibody.
Use: Our Rac1 G15A Agarose Beads are designed to pull down only the active form of Rac1-GEF.
Description: Our Rac1 G15A Agarose beads, in color, are easy to visualize, minimizing potential loss during washes and aspirations of active Rac-GEF pulldown.
Purity and Activity: Purity >90% by SDS-PAGE and Coomassie blue staining. Specifically interacts and precipitaes active Rac-GEF from cell lysate.
Concentration: 800 μL of 50% Agarose slurry, 1 mg/mL Rac1 G15A 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 800 µg Rac1 G15A Agarose Beads (Active Rac-GEF) 1 x 800µg
FunctionPlasma membrane-associated small GTPase which cycles between active GTP-bound and inactive GDP-bound states. In its active state, binds to a variety of effector proteins to regulate cellular responses such as secretory processes, phagocytosis of apoptotic cells, epithelial cell polarization and growth-factor induced formation of membrane ruffles. Rac1 p21/rho GDI heterodimer is the active component of the cytosolic factor sigma 1, which is involved in stimulation of the NADPH oxidase activity in macrophages (By similarity). Essential for the SPATA13-mediated regulation of cell migration and adhesion assembly and disassembly.
Isoform B has an accelerated GEF-independent GDP/GTP exchange and an impaired GTP hydrolysis, which is restored partially by GTPase-activating proteins. It is able to bind to the GTPase-binding domain of PAK but not full-length PAK in a GTP-dependent manner, suggesting that the insertion does not completely abolish effector interaction.
Tissue specificityIsoform B is predominantly identified in skin and epithelial tissues from the intestinal tract. Its expression is elevated in colorectal tumors at various stages of neoplastic progression, as compared to their respective adjacent tissues.
Sequence similaritiesBelongs to the small GTPase superfamily. Rho family.
DomainThe effector region mediates interaction with DEF6.
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 localizationCell membrane. Melanosome. Inner surface of plasma membrane possibly with attachment requiring prenylation of the C-terminal cysteine (By similarity). Identified by mass spectrometry in melanosome fractions from stage I to stage IV.
- Information by UniProt
- Cell migration inducing gene 5 protein
- Cell migration-inducing gene 5 protein
Active Tiam-1 in 2 mg of MDA-231 lysate was pulled down with Rac1 G15A agarose beads and probed with anti-Tiam1 antibody according the assay protocol.
293 cells were transfected with active Tiam1 (ΔN Tiam1). Active Tiam1 in lysate was pulled down with Rac1 G15A agarose beads. Lane 1, Mock Transfection Control. Lane 2, ΔN Tiam1 Transfection.
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.
ab211184 has not yet been referenced specifically in any publications.