GEF and GAP Exchange Assay FAQs

Tip 2: RhoGEF exchange assay biochem kit (cat# BK100)

The RhoGEF exchange assay has been specifically designed for use with 384 well plates.  Below are some important technical tips to optimize the kit for HTS assays. 

Exchange assay reagents
1) This kit contains sufficient purified GTPases and mant-GTP to carry out more than 20 reactions (100 μl volume for 96-well plate) for each GTPase. This corresponds to 60 GEF assays for all three GTPases (Cdc42, RhoA, Rac1) of 100 μl volume using a black flat-bottom 96-well half area plate (Corning plate Cat# 3686). Up to 130 reactions per GTPase can be achieved if a 384-well black round bottom plate (Corning Cat# 3676) is used. This type of 384 well plate is strongly recommended due to the greater signal/noise ratio. DO NOT USE CLEAR PLATES SINCE THAT WILL GIVE YOU SIGNIFICANT BACKGROUND NOISE.

2) The positive control protein, DH/PH domain of human Dbs (Cat # GE01), contained in the kit provides sufficient reagent for at least 20 control assays in a half area 96-well plate format and 130 control assays for a 384-well black round bottom plate format in this assay. Human Dbs is a promiscuous exchange factor for Cdc42 and RhoA, and a weak exchanger on Rac1.

3) The three best characterized small GTPases RhoA, Rac1 and Cdc42 are contained in this kit. If you need to examine more GTPases, a wide selection of small GTPases (Ras, Ran, RhoC and a growing number of GEFs) are available from Cytoskeleton Inc. and can be purchased separately.

4) Many of the reagents in this kit require reconstitution and division into convenient experiment sized aliquots. It is important to carry out the aliquoting step as multiple freeze/thaw cycles of some reagents (for example exchange buffer and purified protein) may result in the inactivation of the reagents. It is strongly recommended to store the exchange buffer without exposing to light for long period of time.

Assay Optimization
The exchange assay kit has been developed to provide a good general substrate for a broad range of research for characterization and examination of guanine nucleotide exchange factors (GEFs). For example, using this kit as outlined in instructions (see manual)  will result in a Vmax value of above 1.8 x 10^-3 mol mant-GTP loaded per mol Cdc42 per second, which is consistent with published data. The exchange assay should be performed at 20°C. A higher temperature may cause a higher intrinsic rate thereby influencing signal to noise ratio significantly. It should be noted, however, that optimization of the exchange assay may be needed for any given GEFs.

There are several parameters that may particularly affect GEF protein activity:

1) Temperature. An exchange reaction at 20°C is required. Besides the fact that lower or higher temperature may cause significant change on signal to noise ratio, different GEFs may require a different optimal temperature for their normal in vitro exchange activity.

2) Protein concentration. A titration of the GEF of interest should be performed to achieve optimal results.

3) Exchange buffer conditions. Although the condition of reaction buffer in this kit has been optimized, it could be necessary to optimize a particular GEF assay by adjusting the salt concentrations (25-500 mM) and the pH (6.0-8.3) using appropriate buffers such as MES, PIPES and Tris.

4) Control reactions. It is important to include control reactions in the assay, particularly if your GEF of interest is in an impure state.

Instrumentation
The fluorescence reaction is based upon an excitation at 360 nm and an emission at 440 nm. Your fluorimeter should therefore be set at an excitation filter wavelength at 360 nm and emission filter wavelength at 440 nm for readings. The bandwidth of the filter should be no more than 20 nm or you may experience significant background noise and reduced sensitivity of the assay. The fluorimeter should be at 20°C and set on kinetic mode.  It is recommended to take a reading once every 30 seconds for at least 60 cycles (30 minutes total). There is no need to elect a blank well, as the reaction without GEFs will serve as a background control.

Tip 3: RhoGAP assay biochem kit (cat# BK105)

The RhoGAP assay has been specifically designed for use with 384 well plates.  Below are some important technical tips to optimize the kit for HTS assays.

GAP assay reagents
1) All protein samples and buffers must be free from phosphate prior to beginning the assays.  If your proteins are in PBS buffer then they must be dialyzed twice in 1000 volumes of 50 mM PIPES pH 7.0 or 50 mM Tris pH 7.5 based buffer to reduce the phosphate content to non
detectable levels.

2) This kit contains sufficient purified GTPases to perform 20 reactions (40 μl volume for 96- well plate) for each GTPase. This corresponds to 80 GAP assays for all four GTPases (Ras, RhoA, Rac1 and Cdc42) using the half area 96-well plate (Corning plate Cat. # 3696). Up to
40 reactions per GTPase can be achieved if a 384-well plate is used.

3) The positive control protein, Rho GAP domain of human p50 RhoGAP (Cat. # GAS01), contained in the kit provides sufficient reagent for at least 12 control assays in a half area 96-well plate format and 24 control assays for a 384-well plate. Human p50 RhoGAP is predominantly a GAP of Rho sub-family members and it is less active at promoting Ras subfamily members GTPase activity.

4) Four well-characterized small GTPases Ras, RhoA, Rac1 and Cdc42 are contained in this kit.  If you need to examine more GTPases, a wide selection of small GTPases (including Ran and RhoC) are available from Cytoskeleton Inc. and can be purchased separately.

5) Some of the reagents in this kit require reconstitution and division into convenient experiment sized aliquots. It is important to carry out the aliquoting step as multiple freeze/thaw cycles of some reagents (for example the purified proteins) will result in the
inactivation of the reagents.

Assay Optimization
The GAP assay kit has been developed to provide good general conditions for a broad range of GTPase activating proteins (GAPs). Using this kit as outlined in the introduction (see manual) will result in an enhanced GTPase activity for RhoA of approximately 10 fold over the endogenous rate which is consistent with published data. The GAP assay should be performed at 37°C. It should be noted however that optimization of the GAP assay may be needed for any given GAP.  And please refer to the trouble shooting guide at the end of the manual for other possible improvements in assay design.

There are several parameters that may particularly affect GAP protein activity:

1) Temperature. An incubation temperature of 37°C is recommended. Different GAPs may require a different optimal temperature for their normal in vitro GAP activity, so it is a good idea to test 20 and 30°C. The lower temperatures may help situations where proteases are
present and they are denaturing proteins at 37ºC.

2) Protein concentration. A titration of the GAP of interest should be performed to achieve optimal results.

3) Reaction buffer conditions. It may be necessary to optimize the assay for a particular GAP by adjusting the salt concentration (25-500 mM), the pH (6.0-8.5) using appropriate buffers such as MES, PIPES and Tris and the MgCl₂ concentration (0.01, 0.1, 1 and 10 mM).

4) Control reactions. It is important to include control reactions in the assay, particularly if your GAP of interest is in an impure state. Control reactions should be small G-protein only and GAP protein only.

Instrumentation
This absorbance based assay is based upon a wavelength of 640 to 660 nm with an optimal of 650 nm. Wavelengths outside of this range are not recommended.