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

Product Uses Include

• Rho signaling pathway studies
• Rho activation assays with primary cells
• Studies of Rho activators and inactivators
• Rho activation assays with limited material
• High throughput screens for Rho activation
  
  

Introduction
This G-LISA™ Rho activation assay measures the levels of GTP-loaded RhoA in cells. The level of activation is measured with luminometry. The G-LISA Rho activation assays are ELISA based Rho activation assays with wich you can measure Rho activity in cells in less than 3 h. For a more detailed introduction on G-LISA™ assays and a listing of other available G-LISA™ kits, see our main G-LISA™ page. For a kit to measure RhoA activation with colorimetric detection, see Cat. # BK124

Kit contents
The kit contains sufficient reagents to perform 96 Rho activation assays. Since the Rho-GTP affinity wells are supplied as strips and the strips can be broken into smaller pieces, each kit can be used for anywhere from one to multiple assays. The following components are included in the kit:

1. 96 Rho-GTP affinity wells
2. Lysis buffer
3. Binding buffer
4. Antigen presenting buffer
5. Wash buffer
6. Antibody dilution buffer
7. Anti-RhoA antibody
8. HRP-labeled secondary antibody
9. Positive control RhoA protein
10. Protease inhibitor cocktail (Cat. # PIC02)
11. Luminescence detection reagents
12. Precision Red™ Advanced protein assay reagent (Cat. # ADV02)
13. Manual with detailed protocols and extensive troubleshooting guide
    
    

Equipment needed

1. Luminometer capable of reading a 96-well plate
2. Multichannel or multidispensing pipettor
3. Orbital microplate shaker capable of at least 200 rpm shaking (400 rpm is optimal)
   
   

Example results
Serum starved Swiss 3T3, HeLa and A431 cells were stimulated with the Rho activating compound lysophosphatidic acid and RhoA activation was measured with BK121 (Fig 1)

	Figure 1. Rho activation by lysophosphatidic acid (LPA) measured by G-LISA™ kit BK121. Swiss 3T3 (mouse), A431 (human) and HeLa (human) cells were serum starved followed by stimulation by LPA.  25 µg of lysates were subjected to the G-LISA™ assay.  Data shown are relative luminescence units (RLU) over background signal (wells incubated with lysis buffer alone instead of cell lysates).  Numbers above LPA bars correspond to fold activation compared to the control serum starved samples.

Go to main G-LISA™ page

G-LISA™ Products:
Cdc42 G-LISA™ Activation Assay, colorimetric format (Cat.# BK127)
Rac1 G-LISA™ Activation Assay, luminescence format (Cat.# BK126)
Rac1,2,3 G-LISA™ Activation Assay, colorimetric format (Cat.# BK125)
RhoA G-LISA™ Activation Assay, colorimetric format (Cat.# BK124)

Associated Products:
Anti-Cdc42 monoclonal antibody (Cat.# ACD03)
Anti-Rac1 monoclonal antibody (Cat.# ARC03)
Anti-RhoA monoclonal antibody (Cat.# ARH03)

For product Datasheets and MSDSs please click on the PDF links below.   For additional information, click on the FAQs tab above or contact our Technical Support department at tservice@cytoskeleton.com

• For our G-LISA Data Analysis (Luminescence) Excel Template please download here.



Product Description

MSDS

Datasheet

Cat #

G-LISA RhoA Activation Assay Biochem Kit (luminescence format) BK121

Moniz et al., 2013. Loss of WNK2 expression by promoter gene methylation occurs in adult gliomas and triggers Rac1-mediated tumour cell invasiveness. Hum. Mol. Genet. 22, 84-95.

Nobe et al., 2012. Two distinct dysfunctions in diabetic mouse mesenteric artery contraction are caused by changes in the Rho A–Rho kinase signaling pathway. E. J. Pharmacol. 683, 217-225.

Wang et al., 2012. RhoA/ROCK-dependent moesin phosphorylation regulates AGE-induced endothelial cellular response. Cardiovascular Diabetology. 11, 7.

Zuo et al., 2012. Cdc42 negatively regulates intrinsic migration of highly aggressive breast cancer cells. J. Cell. Physiol. 227, 1399-1407.

Alvarez et al., 2010. Failure of Bay K 8644 to induce RhoA kinase-dependent calcium sensitization in rabbit blood vessels. British J of Pharmacology. 160 ,1326-37.

Heckman-Stoddard et al., 2009. Haploinsufficiency for p190B RhoGAP inhibits MMTV-Neu tumor progression. Breast Cancer Research. 11 ,http://breast-cancer-research.com/content/11/4/R61.

Hammar et al., 2009. Role of the Rho-ROCK (Rho-Associated Kinase) Signaling Pathway in the Regulation of Pancreatic β-Cell Function. Endocrinology. 150, 2072-2079.

Chastre et al., 2009. TRIP6, a novel molecular partner of the MAGI-1 scaffolding molecule, promotes invasiveness. FASEB J. 23, 916–928.

Kinoshita et al., 2008. Mol. Biol. Cell. 19, 2289

Moniz et al., 2008. WNK2 modulates MEK1 activity through the Rho GTPase pathway. Cell Signal. 20, 1762-68.

Lesato et al., 2008. Tiotropium Bromide Attenuates Respiratory Syncytial Virus Replication in Epithelial Cells. Respiration. 76, 434-441.

Kinoshita et al., 2008. Apical Accumulation of Rho in the Neural Plate Is Important for Neural Plate Cell Shape Change and Neural Tube Formation. Mol. Biol. Cell. 19, 2289-2299.

Scott et al., 2007. J Invest Dermatol. 127, 668.

Schreibelt et al., 2007. Reactive oxygen species alter brain endothelial tight junction dynamics via RhoA, PI3 kinase, and PKB signaling. FASEB J. 21, 3666-3676.

Tanaka et al., 2007. Neural Expression of G Protein-coupled Receptors GPR3, GPR6, and GPR12 Up-regulates Cyclic AMP Levels and Promotes Neurite Outgrowth. J. Biol. Chem. 282, 10506-10515.

Higashibata et al., 2006. BMC Biochem. 7, 19

Zuo et al., 2006. Biochem Biophys Res Commun. 351, 361

Woods and Beier, 2006. J Biol Chem. 281, 13134

Question 1:  There is less than a 2-fold difference in signal intensity between my positive control and lysis buffer blank.  Why?

Answer 1:  To accurately measure luminescence signal intensity between the positive control and buffer blank, please check the instrument settings on the luminometer (see below for suggestions).  We also recommend running some “set-up” experiments with just the buffer blank and positive control to determine optimal settings for detecting the positive control signal 3-5 fold higher than the buffer blank.  It is also important to remember to use a fresh control protein tube for each run of positive control samples.  Do not store and re-use the positive control.

Machine Settings

 Gain    

Gain controls the sensitivity of the machine.  Most luminometers do not allow manual alteration of gain and use an auto-calibration or limited calibration function.  Turn off auto-settings and auto-calibration to use the machine in manual mode.  It is important to contact the luminometer manufacturer or consult the user’s manual to determine the best way to alter the machine sensitivity.  If gain can be altered, one should read at low, medium and high gains to determine the reading within the linear range of the assay (positive control should be 3-5X higher than blank). Gain range varies with instrument.  For example, gain in the Tecan GmbH SpectroFluor Plus ranges from 0 - 150 (where 150 is the highest).

 Integration Time

This parameter can be varied on most machines.  It is a good idea to set the machine at the lowest integration time (usually 10 – 100 ms). Integration times greater than 200 ms are likely to read out of the linear range of the assay and may require lowering of gain or dilution of primary and/or secondary antibodies.

 Shaking

Most machines give the shaking option.  The recommended setting is 5 sec shake, medium orbital speed before read.  This option is not essential to the assay.

 Temperature

Room temperature

 Plate type

Any setting that specifies 96 well flat, white will be sufficient.

 Filters

Luminescence does not require excitation or emission filters so the filter spaces should be left blank.  If this is not an option, excitation can be set at any value and emission should be set between 400-500nm, with 430-445 as optimal setting.

Question 2:  My arbitrary luminescence units (ALU) or relative luminescence units (RLU) are very different from what is depicted in the manual.  Why?

Answer 2:  This is very typical as the luminescence units will vary from luminometer to luminometer based on the machine’s sensitivity and instrument settings.  The important information to take note of is what the relationship is between buffer blank and positive control luminescence values.  The positive control signal should be 3-5 fold greater than the buffer blank luminescence signal.  If that is the case, then the G-LISA assay is functioning in the linear range and experimental samples can now be processed.

If you have any questions concerning this product, please contact our Technical Service department at tservice@cytoskeleton.com.