RhoA Pulldown Activation Assay Kit

Product Uses Include

• Analysis of in vivo RhoA activation

Introduction
The Rho switch operates by alternating between an active, GTP-bound state and an inactive, GDP-bound state. Understanding the mechanisms that regulate activation / inactivation of the GTPases is of obvious biological significance and is a subject of intense investigation. The fact that many Rho family effector proteins will specifically recognize the GTP bound form of the protein has been exploited experimentally to develop a powerful affinity purification assay that monitors RhoA protein activation. The assay uses the Rho binding domain (RBD) of the Rho effector protein, Rhotekin. The RBD motif has been shown to bind specifically to the GTP-bound form of RhoA. The fact that the RBD region of Rhotekin has a high affinity for GTP-RhoA and that Rhotekin binding results in a significantly reduced intrinsic and catalytic rate of GTP hydrolysis make it an ideal tool for affinity purification of GTP-RhoA from cell lysates. The Rhotekin-RBD protein supplied in this kit contains Rhotekin residues 7-89 and is in the form of a GST fusion protein, which allows one to "pull-down" the Rhotekin-RBD/Rho-GTP complex with brightly colored glutathione affinity beads. The assay therefore provides a simple means of quantitating RhoA activation in cells. The amount of activated RhoA is determined by a western blot using a RhoA specific antibody.

Kit contents
The kit contains sufficient materials for 80 assays depending on activation levels of Rho in cells and includes reagents for positive and negative controls. The following components are included:

1. GST-tagged Rhotekin-RBD protein on colored agarose beads (Cat. # RT02)
2. RhoA-specific monoclonal antibody (Cat. # ARH03)
3. His-tagged RhoA protein (Cat. # RH01)
4. GTP?S: (non-hydrolyzable GTP analog) (Cat. # BS01)
5. GDP
6. Cell lysis Buffer
7. Wash Buffer
8. Loading Buffer
9. STOP Buffer
10. Protease inhibitor cocktail (Cat. # PIC02)
11. Manual with detailed protocols and extensive troubleshooting guide

Figure 1. The brightly colored glutathione agarose beads in BK036 makes the kit easy to use.

Equipment needed

1. SDS-PAGE minigel system and western blotting transfer apparatus
   
   

Example results
The RhoA activation assay was tested by loading the RhoA protein in cell lysates with either GTP?S or GDP. As expected, the GTP?S-loaded RhoA is very efficiently precipitated while very little GDP-loaded RhoA is precipitated (Fig. 2).

Figure 2. Results from BK036 RhoA activation assay. Activated Rho was precipitated and detected in a Western blot using kit BK036. The first lane shows a 50 ng recombinant His-tagged RhoA standard (Rec. His-RhoA). The following lanes shows the pull-down of inactive, GDP-loaded RhoA (RhoA-GDP PD) or active, GTP?S-loaded RhoA (RhoA-GTP PD) from equal amounts of cell lysates.

Please check out the new version of the Rac Activation Assay and associated products:

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)
RhoA G-LISA Activation Assay, luminescence format (Cat.# BK121)

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

Question 1: I have high background and/or multiple bands on my western blot.  How can I fix this?

Answer 1:  There are multiple causes of high background and/or multiple bands.  Some suggestions to improve background signal include:

1.   When blotting use 70v for 45min only as the small G-proteins are very mobile.
2. Fully remove SDS from the gel by using a non-SDS containing buffer for transfer and performing a full 15 min gel wash step in the transfer buffer before blotting.
3. Dry the PVDF membrane for 30 min after transfer and before blocking (not necessary for nitrocellulose)
4. Making sure that the TBST contains 10 mM Tris, 0.05% Tween 20 and 150 mM NaCl.
5. Incubating with the primary antibody overnight at 4°, C and using the appropriate ECL detection system. 

Question 2: How much of the beads should I use for my pull-down experiments?

Answer 2:  Rhotekin-RBD beads (Cat. # RT02) will bind to Rho-GDP with a much lower affinity than Rho-GTP.  If too many rhotekin-RBD beads are added to the pull-down assay there will be significant binding to inactive (GDP-bound) RhoA.  The result of this will be an underestimation of RhoA activation.  For this reason, we highly recommend performing a bead titration to determine optimal conditions for any given RhoA activation or inactivation assay.  Once optimal conditions have been established, bead titrations should no longer be necessary. We recommend 25, 50 and 100 &mu, g bead titrations.

Question 3:  How can I test whether the beads are working properly?

Answer 3:  A standard biological assay for rhotekin-RBD beads consists of a RhoA protein pull-down from cells loaded with either GTP&gamma, S (Cat. # BS01) or GDP.  Here are guidelines to follow (see Cat. # RT02 and BK036 datasheets for more details):

Positive Cellular Protein Control:

Total cell lysate (300 &ndash, 800 &mu, g) should be loaded with GTP&gamma, S as a positive control for the pull-down assay.  The following reaction details how to load endogenous RhoA with the nonhydrolysable GTP analog (GTP&gamma, S).  This is an excellent substrate for rhotekin-RBD beads and should result in a strong positive signal in a pull-down assay.

a) Perform GTP loading on 300 &ndash, 800 &mu, g of cell lysate (0.5 mg/ml protein concentration) by adding 1/10th volume of Loading Buffer.

b) Immediately add 1/100th volume of GTP&gamma, S (200 &mu, M final concentration).  Under these conditions, 5 - 10% of the RhoA protein will load with non-hydrolysable GTP&gamma, S and will be &ldquo, pulled-down&rdquo, with the rhotekin-RBD beads in the assay.

c) Incubate the control sample at 30°, C for 15 min with gentle rotation.

d) Stop the reaction by transferring the tube to 4°, C and adding 1/10th volume of STOP Buffer.

e) Use this sample immediately in a pull-down assay.

Negative Cellular Protein Control:

This reaction should be performed in an identical manner to the Positive Control reaction except that 1/100th volume of GDP (1 mM final concentration) should be added to the reaction in place of the GTP&gamma, S.  Loading endogenous RhoA with GDP will inactivate RhoA and this complex will bind very poorly to rhotekin-RBD beads.

If you have any questions concerning this product, please contact our Technical Service department at infohoelzel.de