Signal Transduction Assays

Apart from Activation Assays, Cytoskeleton provides some innovative kits for studying GEFs and GAPs. In particular the Cat. # BK100 kit is useful for measuring GTP or GDP exchange in Rho family members, and it can also be used to study a new GEF or a new GEF/small G-protein combination. The Rho GAP kit, Cat. # BK105, measures the release of phosphate from GTP hydrolysis by a Rho family protein which is accelerated by the GAP protein provided in the kit.

 

For more information see the datasheets, or contact Technical Support at tservice@cytoskeleton.com .

GTPase_cycle_copy_1

Cytoskeleton's products have been cited hundreds of times over the past 18 years. Please contact Cytoskeleton's Technical Support at tservice@cytoskeleton.com for the latest citations.

 

RhoGAP Assay Biochem Kit (Cat. # BK105)

Zhang et al., 2010. A Novel Testis-specific GTPase Serves as a Link to Proteasome Biogenesis: Functional Characterization of RhoS/RSA-14-44 in Spermatogenesis. Mol. Biol. Cell. 4312–4324.

Rothenfluh et al., 2006. Distinct Behavioral Responses to Ethanol Are Regulated by Alternate RhoGAP18B Isoforms. Cell. 127, 199–211.

 

RhoGEF Assay Biochem Kit (Cat. # BK100)

Moey et al., 2011. Ginseng (Panax quinquefolius) attenuates leptin-induced cardiac hypertrophy through inhibition of p115RhoGEF-RhoA/ROCKdependent MAPK pathway activation. J. Pharmacol. Exp. Ther. DOI:10.1124/jpet.111.182600.

Kim et al., 2009. Dipeptidyl Peptidase IV Inhibition With MK0431 Improves Islet Graft Survival in Diabetic NOD Mice Partially via T-Cell Modulation. Diabetes. 58, 641-651.

Li et al., 2009. PLC-gamma1 and Rac1 Coregulate EGF-Induced Cytoskeleton Remodeling and Cell Migration. Mol. Endocrinol. 23, 901-913.

Question 1:  I'm expecting to see only a small difference in RhoA activation.  What is the best way to measure this and what sensitivity can I expect?

Answer 1:  To detect small differences in RhoA activation between control and treated conditions, we recommend either the absorbance or luminescence-based RhoA G-LISA activation assays (Cat. # BK124 or BK121, respectively).  The absorbance-based RhoA G-LISA (Cat. # BK124) has a detection limit of 0.050 ng activated RhoA and a linear detection range of 0.05 – 2 ng activated RhoA.  The coefficient of variation (8 replicates) is 12%.  The luminescence-based RhoA G-LISA (Cat. # BK121) is recommended for use with 3-D cultures grown in matrigel or collagen matrices because of the additional sensitivity this kit offers.  The luminescence-based RhoA G-LISA has a detection limit of 0.025 ng activated RhoA and a linear range of 0.025-1 ng activated RhoA.

 

Question 2:  I have a purified protein that is possibly a Rho effector and a microtubule binding protein.  How do I approach this project?

Answer 2:   A good reference we recommend as a guide for studying such a protein is: Palazzo et al., 2001 (Nat. Cell Bio. mDia mediates Rho-regulated formation and orientation of stable microtubules. 3, 723-729).  In general, domain cloning should be utilized and then the RhoA binding activity of each domain would be measured with the positive control RhoA protein (enough for 96-assays) included in each RhoA GLISA kit (Cat. # BK124).  The presence of effector domain should reduce the RhoA signal because it will compete for RhoA protein in the well.  To examine binding between the purified protein and microtubules, we recommend using the microtubule binding protein spin-down assay biochem kit (Cat. # BK029).

 

For more information, click on the Documents tab above and see the datasheet, or contact Technical Support at tservice@cytoskeleton.com

  1. Arf1 G-LISA Activation Assay Kit (Colorimetric Based) - 96 assays BK132

    G-LISA Arf1 Activation Assay Biochem Kit (Colorimetric Based)

    Learn More
  2. Cdc42 G-LISA Activation Assay (Colorimetric format) - 24 assays BK127-S

    G-LISA Cdc42 Activation Assay Biochem Kit (Colorimetric format)

    Learn More
  3. Cdc42 G-LISA Activation Assay (Colorimetric format) - 96 assays BK127

    G-LISA Cdc42 Activation Assay Biochem Kit (Colorimetric format)

    Learn More
  4. Rac1 G-LISA Activation Assay (Luminescence format) - 96 assays BK126
    G-LISA Rac 1 Activation Assay Biochem Kit (luminescence format)
    Learn More
  5. Rac1,2,3 G-LISA Activation Assay (Colorimetric format) 96 assays BK125
    G-LISA Rac 1,2,3 Activation Assay Biochem Kit (colorimetric format)
    Learn More
  6. Ras G-LISA Activation Assay Kit (Colorimetric Based) - 96 assays BK131
    G-LISA Ras Activation Assay Biochem Kit (Colorimetric format) Learn More
  7. RhoA G-LISA Activation Assay (Luminescence format) 96 assays BK121
    G-LISA RhoA Activation Assay Biochem Kit (luminescence format)
    Learn More
  8. RhoA G-LISA Activation Assay Kit (Colorimetric format) 24 assays BK124-S

    G-LISA RhoA Activation Assay Biochem Kit (Colorimetric format)

    Learn More
  9. RhoA G-LISA Activation Assay Kit (Colorimetric format) 96 assays BK124
    G-LISA RhoA Activation Assay Biochem Kit (colorimetric format)
    Learn More
  10. Signal-Seeker™ Acetyl-Lysine Detection Kit (10 assay) BK163-S
    Signal Seeker Acetylation Detection Kit (10 assays, immunoprecipitation format) Learn More
  11. Signal-Seeker™ Acetyl-Lysine Detection Kit (30 assay) BK163
    Signal Seeker Acetylation Detection Kit (30 assays, immunoprecipitation format) Learn More
  12. Signal-Seeker™ Phosphotyrosine Detection Kit (10 assay) BK160-S
    Signal Seeker Phosphotyrosine Detection Kit (10 assays, immunoprecipitation format) Learn More
  13. Signal-Seeker™ Phosphotyrosine Detection Kit (30 assay) BK160
    Signal Seeker Phosphotyrosine Detection Kit (30 assays, immunoprecipitation format)
    Learn More
  14. Signal-Seeker™ SUMOylation 1 Detection Kit (10 assay) BK165-S
    Signal Seeker SUMOylation 1 Detection Kit (10 assays, immunoprecipitation format) Learn More
  15. Signal-Seeker™ SUMOylation 1 Detection Kit (30 assay) BK165
    Signal Seeker SUMOylation 1 Detection Kit (30 assays, immunoprecipitation format) Learn More
  16. Signal-Seeker™ SUMOylation 2/3 Detection Kit (10 assay) BK162-S
    Signal Seeker SUMOylation 2/3 Detection Kit (10 assays, immunoprecipitation format) Learn More
  17. Signal-Seeker™ SUMOylation 2/3 Detection Kit (30 assay) BK162
    Signal Seeker SUMOylation 2/3 Detection Kit (30 assays, immunoprecipitation format) Learn More
  18. Signal-Seeker™ Ubiquitination Detection Kit (10 assay) BK161-S
    Signal-Seeker Ubquitination Detection Kit (10 assays, immunoprecipitation format) Learn More
  19. Signal-Seeker™ Ubiquitination Detection Kit (30 assay) BK161
    Signal-Seeker Ubquitination Detection Kit (30 assays, immunoprecipitation format) Learn More