Rhotekin RBD Agarose Beads (ab211178) is part of the reagents, controls & accessories range. Abcam offers high-quality biological reagents and tools including antibodies, proteins, assays, cell lines and lysates.
Images
Key facts
- Form
- Liquid
Recommended products
Rhotekin RBD Agarose Beads (ab211178) is part of the reagents, controls & accessories range. Abcam offers high-quality biological reagents and tools including antibodies, proteins, assays, cell lines and lysates.
Key facts
- Form
- Liquid
- Storage buffer
Constituents: 50% Glycerol (glycerin, glycerine)
Storage
- Shipped at conditions
- Blue Ice
- Appropriate short-term storage conditions
- -20°C
- Appropriate long-term storage conditions
- -20°C
Notes
Use: Our Rhotekin RBD Agarose beads are designed to pull down only the active form of Rho.
Description: Our Rhotekin RBD Agarose beads are colored for easy visualization, minimizing potential loss during washes and aspirations during Rho-GTP pulldown.
Activity: Product specifically interacts and precipitates GTP-bound Rho from cell lysate.
Concentration: 800 μL of 50% Agarose slurry, 400 μg Rhotekin RBD 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.
Supplementary info
- This supplementary information is collated from multiple sources and compiled automatically.
- Activity summary
Rhotekin also known as RTKN is a scaffold protein with a molecular mass of approximately 62 kDa. It interacts with Rho GTPases facilitating the assembly of signaling complexes. Expressed in various tissues RTKN displays high levels of expression in liver and kidney tissues. Alternative splicing of RTKN results in multiple transcript variants.
- Biological function summary
RTKN modulates several cellular processes by influencing cytoskeletal organization. This protein is a part of a Rho GTPase signaling complex that plays roles in cellular morphology and movement. It binds to activated Rho which leads to inhibition of Rho-associated kinase (ROCK) an important regulator of actin structures. This binding stabilizes RTKN's influence on cytoskeletal arrangement.
- Pathways
Cellular processes worldwide involve Rho GTPase signals where RTKN plays an important role. It links to the Wnt signaling pathway pivotal for cell proliferation and differentiation through its interaction with Dishevelled proteins. RTKN functions alongside related proteins like ROCK within these pathways. These interactions influence signal transduction and cellular responses.
- Associated diseases and disorders
RTKN shows associations with various cancers including gastric cancer through its interactions with aberrant Rho GTPase activity. Moreover RTKN has implications in neurodegenerative diseases like Alzheimer's often involving disrupted Rho signaling alongside proteins like tau. These connections highlight RTKN's importance in disease progression and make it a potential therapeutic target.
Product promise
We are dedicated to supporting your work with high quality reagents and we are here for you every step of the way should you need us.
In the unlikely event of one of our products not working as expected, you are covered by our product promise.
Full details and terms and conditions can be found here:
Terms & Conditions.
1 product image
Functional Studies - Rhotekin RBD Agarose Beads (ab211178)
Lane 1, GTPase Immunoblot Positive Control. Lane 2, MDA-231 cell lysate loaded with GDP and incubated with Rhotekin RBD Agarose beads. Lane 3, 293 cell lysate loaded with GTPγS and incubated with Rhotekin RBD Agarose beads.
Downloads
Product protocols
- Visit the general protocols
- Visit troubleshooting
Please note: All products are 'FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC OR THERAPEUTIC PROCEDURES'.
For licensing inquiries, please contact partnerships@abcam.com