Enabling Epigenetics Research

Ras GTPase Chemi ELISA Kit
Catalog No. 52097(Version C1)

Copyright 2021 Active Motif, Inc.

Revision

Date

Description of Change

C1

August, 2024

Formatting update

Information in this manual is subject to change without notice and does not constitute a
commitment on the part of Active Motif, Inc. It is supplied on an “as is” basis without any
warranty of any kind, either explicit or implied. Information may be changed or updated inthis manual at any time.
This documentation may not be copied, transferred, reproduced, disclosed, or duplicated,
in whole or in part, without the prior written consent of Active Motif, Inc. This documentation
is proprietary information and protected by the copyright laws of the United States andinternational treaties.
The manufacturer of this documentation is Active Motif, Inc.
© 2021 Active Motif, Inc., 1914 Palomar Oaks Way, Suite 150; Carlsbad, CA, 92008.
All rights reserved.
All trademarks, trade names, service marks or logos referenced herein belong to theirrespective companies.

activemotif.com

Contents
Overview  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Flow Chart of Process . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  2
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  3
Kit Performance and Benefits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  4
Kit Components and Storage  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  5
Additional Materials Required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  5
Protocols  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  6
Buffer Preparation and Recommendations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  6
Quick Chart for Preparing Buffers  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  8
Preparation of Whole-Cell Extract  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  9
Ras GTPase Chemi ELISA Kit  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
Troubleshooting Guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Technical Services . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

activemotif.com

Overview
Small GTP-binding proteins (GTPases) are important regulators of signal transduction
pathways. The small GTPase Ras acts as a key regulator of cellular functions including
proliferation and differentiation and is also implicated in tumorigenesis, tumor invasion andmorphogenesis. Oncogenic mutations in the ras gene are present in approximately 30% of
all human cancers1. Because of the critical role of Ras in tumor development, it is important
to be able to screen novel signaling pathways for activating Ras. Traditional methods formonitoring Ras activation, such as Western blotting, are tedious and time consuming and notsuitable to high-throughput analysis.
The Ras GTPase Chemi ELISA Kit is designed specifically for the study of Ras activation andcan be used to study novel signaling pathways for activating Ras or for detecting oncogenic
Ras. Ras GTPase ELISA Kits contain a Raf-RBD protein fused to GST that will be coatedonto the provided 96-well, glutathione-coated plate. The activated Ras contained in thecellular extract specifically binds to Raf-RBD, while inactive Ras does not bind. Bound Ras is
detected by incubating with a primary antibody that detects H-Ras in mouse and H- & K-Ras
in human samples. Addition of a secondary antibody conjugated to horseradish peroxidase(HRP) and developing solution provides a sensitive chemiluminescent readout that is easily
quantified by luminescence. The 96-well plate is suitable for manual use or high-throughput
screening applications.
Product

FormatCatalog No.

Ras GTPase Chemi ELISA Kit

1 x 96 rxns

52097

activemotif.com|

1Flow Chart of Processactivemotif.com|

2

Introduction
Ras GTPase
GTPases (also called GTP-binding proteins) are a family of enzymes that bind to andhydrolyze GTP, allowing them to function as molecular switches. When bound to GDP, theGTPase protein is in its inactive form. Activation is controlled by regulatory proteins called
guanine nucleotide exchange factors (GEFs), which induce the release of GDP. Because GTP
is present in the cell in a large excess over GDP, the resulting empty nucleotide-binding site
is filled by GTP and the GTPase is activated. Another class of proteins, GTPase-activating
proteins (GAPs), speed up hydrolysis of GTP to GDP, inactivating the GTPase . The figure
below illustrates Ras activation.

Activating
signal
Pi

GAPs
H2O

Ras-GDP
Ras-GTP

+

GTP

GEFs
GDP

Effectors
Cellular responses
The small GTPase Ras family regulates a variety of cell functions including proliferation anddifferentiation. Family members include Ras (H, K, N, R, M, and TC21), Rap (1A, 1B, 2A, and
2B) and Ral (A and B), and are characterized by similarities in their effector domains3. Ras
proteins consist of about 190 amino acid residues that are highly conserved in the N and C
termini. Most variations between proteins occur near the C-terminal hypervariable domain,
and this variation is presumed to be responsible for differences in function4.
Activated Ras in turn activates several distinct effectors, such as the serine-threonine kinase
Raf1, phosphoinositide 3´-kinase (PI3K) and RalGDS. One of the best characterized effector
molecules activated by Ras is Raf kinase. Activation of Raf initiates a phosphorylationcascade involving MEK and ERK protein kinases leading to the activation of transcription
factors like Elk1.
Normally, Ras-signaling cascades are only transiently activated because GTPase’s intrinsic
hydrolyzing activity gradually converts GTP to GDP. This conversion is also enhanced by
the presence of GAP proteins. However, there are mutant oncogenic Ras proteins thatremain constitutively in the active GTP-bound form. Identified mutations are limited to asmall number of sites that abolish GAP-induced hydrolysis of GTP, resulting in continuous
stimulation of cellular proliferation. Oncogenic mutations in the ras gene are present inapproximately 30% of all human cancers. Colon and pancreatic cancers have mutations in
the K-ras gene, urinary tract and bladder cancers have mutations in the H-ras gene, andmutations in N-ras are associated with leukemia4.

activemotif.com|

3Kit Performance and Benefits
The Ras GTPase ELISA Kit is for research use only. Not for use in diagnostic procedures.
Detection limit: > 3 µg whole-cell extract/well or > 0.6 ng purified protein/well.
Range of Detection: The Ras GTPase Chemi ELISA can give relative quantitation of RasGTPase when used in the range of 3 to 25 µg of cell extract/well and compared to theprovided control HeLa + EGF cell extract. (See graph below.)
Cross-reactivity: Ras GTPase ELISA specifically detects activated H- and K-Ras in human and
H-Ras in rodent samples.
Assay time: 4.5 hours.

3000000
Activation (RLU)

2500000

2000000

293 (Ras -ve)
HeLa unstimulated
HeLa + EGF

1500000

1000000
500000
0

3.13

6.25

12.5
25
Extract (µg/well)50

Quantification of activated Ras: Increasing amounts of whole-cell extracts from unstimulated
293T/17 and EGF stimulated HeLa cells were assayed for Ras activity using the Ras GTPase Chemi
ELISA Kit. To illustrate the Kit’s specificity for activated Ras, 293T/17 cells which do not contain basal
levels of activated Ras were used as a negative control. Data was also shown for unstimulated HeLa
cells, which do contain basal levels of activated Ras. This data is provided for demonstration only.

activemotif.com|

4Kit Components and Storage
Please store each component at the temperature indicated in the table below upon receiptof the kit. Kit components must be stored at the temperatures listed below for 24 hours prior
to use.
Reagents

Quantity

Storage

Hela whole-cell extract (EGF treated, 2.5 µg/µL)

2 x 40 µL-80°C

GST-Raf-RBD (2 mg/mL)

4 x 25 µL-80°C

H-Ras antibody

11 µL

-20°C

Anti-rat HRP-conjugated IgG (0.25 µg/µL)

11 µL

-20°C

Protease Inhibitor Cocktail (PIC)

500 µL-20°C

Lysis/Binding Buffer AM11

50 mL

4°C

2 x 22 mL

4°C

10X Antibody Binding Buffer AM2

2.2 µL

4°C

Chemiluminescent Reagent

2 mL

4°C

Reaction Buffer

4 mL

4°C

96-well assay plate

1 ea

4°C

Plate sealer

2 ea

4°C

10X Wash Buffer AM2

Additional Materials Required•

Multi-channel pipettor•

Multi-channel pipettor reservoirs•

Rocking platform at room temperature and 4°C•

Microplate luminometer or CCD camera-coupled imaging system•

PBS

activemotif.com|

5

Protocols
Please read the entire protocol before starting.

Buffer Preparation and Recommendations
Preparation of Complete Lysis/Binding Buffer
We provide an excess of Lysis/Binding Buffer in order to perform the assay AND to prepare
customized cell extracts. Prepare the amount of Complete Lysis/Binding Buffer required forthe assay by adding 10 µL of Protease Inhibitor Cocktail per mL of Lysis/Binding Buffer (seethe Quick Chart for Preparing Buffers in this section). Some of the protease inhibitors lose
their activity after 24 hours once diluted. Therefore, we recommend using the Complete
Lysis/Binding Buffer immediately for cell lysis. The remaining amount should be discarded if
not used in the same day.
Preparation of 1X Wash Buffer
Prepare the amount of 1X Wash Buffer required for the assay as follows: For every 10 mL of
1X Wash Buffer required, dilute 1 mL 10X Wash Buffer AM2 with 9 mL distilled water (see the
Quick Chart for Preparing Buffers in this section). Mix gently to avoid foaming. The 1X Wash
Buffer may be stored at 4°C for one week. The Tween 20 contained in 10X Wash Buffer AM2
may form clumps, therefore homogenize the buffer by vortexing for 2 minutes prior to use.
Preparation of 1X Antibody Binding Buffer
Prepare the amount of 1X Antibody Binding Buffer required for the assay as follows: For
every 10 mL of 1X Antibody Binding Buffer required, dilute 1 mL 10X Antibody Binding Buffer
AM2 with 9 mL distilled water (see the Quick Chart for Preparing Buffers in this section)*. Mix
gently to avoid foaming. Discard remaining 1X Antibody Binding Buffer after use. The BSA
contained in the 10X Antibody Binding Buffer AM2 may form clumps, therefore homogenize
the buffer by warming to room temperature and vortexing for 1 minute prior to use.
Diluted Primary Antibody
The primary Ras antibody recognizes H- and K-Ras in human and H-Ras in rodent samples.
The supplied antibody will be diluted 1:500 in 1X Antibody Binding Buffer (see the Quick
Chart for Preparing Buffers in this section). Avoid multiple freeze/thaw cycles.
Diluted HRP-conjugated Secondary Antibody
HRP-conjugated anti-rat IgG is used as the secondary antibody to detect bound primary
antibody. The supplied antibody will be diluted 1:5000 in 1X Antibody Binding Buffer. This
dilution should be made by performing a 1:10 dilution followed by a 1:500 dilution (see the
Quick Chart for Preparing Buffers in this section). Avoid multiple freeze/thaw cycles.

activemotif.com|

6Preparation of Chemiluminescent Working Solution
The Chemiluminescent Reagent and Reaction Buffer should be warmed to room temperature
before use. These components are light sensitive, therefore, we recommend avoiding
direct exposure to intense light during storage. Prior to use, place the Chemiluminescent
Reagent and Reaction Buffer at room temperature for at least 1 hour. In a separate
container, mix 1 volume of Chemiluminescent Reagent with 2 volumes of Reaction Buffer
to prepare the Chemiluminescent Working Solution (see the Quick Chart for Preparing
Buffers in this section). The Chemiluminescent Working Solution is stable for several
hours. After the Chemiluminescent Working Solution is aliquoted into the wells, discard theremaining solution.
GST-Raf-RBD
The GST-Raf-RBD contains a Ras Binding Domain and is used to capture activated Ras on
the glutathione-coated plate. GST-Raf-RBD must be aliquoted into small fractions to avoid
freeze/thaws. Four tubes of the GST-Raf-RBD are provided and must be stored at -80°C
upon receipt.
96-well assay plate
The 96-well assay plate is a solid plate. Determine the appropriate number of wells required
for testing. Cover the unused wells with a portion of the plate sealer while you perform theassay to prevent contamination. The content of the wells is stable at room temperature if
kept dry and, therefore, can be used later for a separate assay. Return the plate to the foil
pouch, seal and store at 4°C between uses.
Extract
The HeLa whole-cell extract (EGF treated) is provided as a positive control for Ras activation.
Sufficient extract is supplied for 8 reactions per plate. This extract is optimized to givea strong signal when used at 25 µg/well. We recommend aliquoting the extract in 21 µL
fractions and storing at -80ºC. Avoid multiple freeze/thaw cycles of the extract.
Note:

The HeLa whole-cell extract (EGF treated) is sensitive to GTP hydrolysis at
4°C, thus we recommend thawing it no more than 15 minutes prior to use.

activemotif.com|

7Quick Chart for Preparing Buffers*
Reagents to prepare

Components

Complete
Lysis/Binding Buffer

Protease inhibitor cocktail

1X Antibody
Binding Buffer**Primary Antibody
1/500 Dilution

1 well(12 wells)(48 wells)(96 wells)

0.55 µL

6.5 µL

26 µL

52 µL

55 µL

643.5 µL

2.574 mL

5.148 mL

Total required

55.55 µL

650 µL

2.6 mL

5.2 mL

Distilled water

112.5 µL

1.26 mL

4.95 mL

9.9 mL10X Ab Binding Buffer AM2

12.5 µL

140 µL

550 µL

1.1 mL

Total required

125 µL

1.4 mL

5.5 mL111 mLRas antibody

0.11 µL

1.3 µL5.2 µL

10.4 µL

1X Ab Binding Buffer

55 µL

648.7 µL

2.6 mL

5.2 mL

55.11 µL

650 µL

2.605 mL

5.210 mL

HRP-conjugated antibody

1 µL

1 µL

1 µL

2 µL

1X Ab Binding Buffer

9 µL

9 µL

9 µL

18 µL

Total required

10 µL

10 µL

10 µL

20 µL

Pre-diluted HRP

0.11 µL

1.3 µL5.2 µL

10.4 µL

1X Ab Binding Buffer

55 µL

648.7 µL

2.6 mL

5.2 mL

Total required

55.11 µL

650 µL

2.605 mL

5.210 mLDistilled water

2.52 mL

30.6 mL

122.4 mL

243 mL

10X Wash Buffer AM2

280 µL

3.4 mL

13.6 mL

27 mL

Total required

2.8 mL34 mL

136 mL

270 mL

Chemiluminescent Reagent

17 µL

217 µL867 µL

1.734 mL

Reaction Buffer

34 µL

434 µL

1.734 mL

3.468 mL

Total required

51 µL

651 µL2.601 mL

5.202 mL

Lysis Buffer AM1

Total required
Secondary Antibody
Pre-Dilution (1/10)

Secondary Antibody
1/500 Dilution

1X Wash Buffer

ChemiluminescentWorking Solution* The Quick Chart includes an excess of components to perform the assay.
** Volumes listed refer to the preparation of buffers for diluting both the primary & secondary antibodies.

activemotif.com|

8Preparation of Whole-Cell Extract
This procedure can be used for a confluent cell layer of 10 cm2 (100 mm dish) or 2 x 107 cells.
1.

Treat the cells as required for Ras activation.

2.

Wash the cells with 5 mL ice-cold PBS (10 mM phosphate buffer, pH 7.5, 150
mM NaCl).

3.

For adherent cells add 500 µL of Complete Lysis/Binding Buffer and scrape cells (witha rubber policeman). For suspension cells resuspend cell pellet in 1 mL Complete
Lysis/Binding Buffer.

4.

Transfer cells to a microcentrifuge tube. Incubate 15 minutes at 4°C.

5.

Vortex tube for 10 seconds and then centrifuge for 10 minutes at 14,000 rpm at 4°C.

6.

Collect the supernatant at 4°C.
10X PBSFor 250 mL, mix:

0.1 M phosphate buffer, pH 7.5

3.55 g Na2HPO4 + 0.61 g KH2PO4

1.5 M NaCl

21.9 g

27 mM KCl

0.5 g

7.

Measure the protein content by a Bradford-based assay.

8.

For best results, extracts should be used immediately in the Ras ELISA.

Adjust to 250 mL with distilled water. Prepare a 1X PBS solution by adding 10 mL 10X PBS to
90 mL distilled water. Sterilize the 1X PBS by filtering through a 0.2 µm filter. The 1X PBS is at
pH 7.5. Store the filter-sterilized 1X PBS solution at 4°C.
OPTIONAL - GTPγS or GDP Treatment
The protocol below is provided as an optional procedure for the production of positive andnegative controls for Ras activation. GTPγS acts as an activator while GDP acts as an inhibitor
to Ras activation. Use > 200 µg of extract for each treatment.
1.

Dilute the test extracts to desired concentration in Complete Lysis/Binding Buffer( >200 µg per well is recommended).

2.

To each tube, add 0.5M EDTA pH 8.0 to a final concentration of 10 mM.

3.

To each tube, add 10 mM GTPγS or 100 mM GDP to a final concentration of 0.1 mM
and 1.0 mM, respectively.

4.

Incubate at 30°C for 15 minutes.

5.

To each tube, add 1M MgCl2 to a final concentration of 60 mM.

6.

Extracts should be used immediately in the Ras ELISA.

activemotif.com|

9

Ras GTPase Chemi ELISA Kit
Determine the appropriate number of microwell strips required for testing samples, controls
and blanks in duplicate. Cover the unused wells with a portion of the plate sealer while you
perform the assay. The content of these wells is stable at room temperature if kept dry and,
therefore, can be used later for a separate assay. Return the plate to the foil pouch, seal andstore at 4°C between uses.
Prepare the Complete Lysis/Binding Buffer, 1X Wash Buffer and 1X Antibody Binding Buffer
as described in the section Buffer Preparation and Recommendations. Multi-channel pipettor
reservoirs may be used for dispensing the Complete Lysis/Binding Buffer, Wash Buffer,
Antibody Binding Buffer and Chemiluminescent Working Solution into the wells being used.
Binding of Ras
Note:

For optimal kit performance, kit components must be stored at therecommended storage temperatures indicated on page 5 of the manual for 24hours prior to use.

1.

Add 2 µg of GST-Raf-RBD diluted in 50 µL of Complete Lysis/Binding Buffer to each well
to be used. (1 µL of GST-Raf-RBD in 49 µL Complete Lysis/Binding Buffer per well).

2.

Use the provided adhesive cover to seal the plate. Incubate for 1 hour at 4°C with mild
agitation (100 rpm on a rocking platform).

3.

Wash each well 3 times with 200 µL 1X Wash Buffer. For each wash, flick the plateover a sink to empty the wells, then tap the inverted plate 3 times on absorbent
paper towels.

4.

Sample wells: Dilute test extracts to desired concentration in Complete Lysis/Binding
Buffer. Sample can be used at 50-200 µL per well, depending on stock concentration.
We recommend using 10-100 µg of extract diluted in Complete Lysis/Binding Buffer
per well.
Positive control wells: Thaw the provided HeLa (EGF treated) extract on ice for no morethan 15 minutes prior to use. Add 25 µg of this extract diluted in 50 µL of Complete
Lysis/Binding Buffer per well (10 µL of extract in 40 µL of Complete Lysis/Binding Buffer
per well).
Blank wells: Add 50 µL Complete Lysis/Binding Buffer only per well.

5.

Cover the plate and incubate for 1 hour at room temperature with mild agitation (100rpm on a rocking platform).

6.

Wash each well 3 times with 200 µL 1X Wash Buffer (as described in Step 3)

Binding of Primary Antibody
7.

Add 50 µL diluted H-Ras antibody (1:500 dilution in 1X Antibody Binding Buffer)to wells.

8.

Cover the plate and incubate for 1 hour at room temperature without agitation.

9.

Wash the wells 3 times with 200 µL 1X Wash Buffer (as described in Step 3).

activemotif.com|

10

Binding of Secondary Antibody
10. Add 50 µL diluted HRP antibody (1:5000 dilution in 1X Antibody Binding Buffer) to allwells being used.
11. Cover the plate and incubate for 1 hour at room temperature without agitation.
12. During this incubation, place Chemiluminescent Reagent and Reaction Buffer atroom temperature.
13. Wash the wells 4 times with 200 µL 1X Wash Buffer (as described in Step 3).
Chemiluminescent Detection
14. Add 50 µL room-temperature Chemiluminescent Working Solution to all wells
being used.
15. Read chemiluminescence using a luminometer or CCD camera system. Readings
should be taken within 15 minutes to minimize changes in signal intensity.

activemotif.com|

11

References
1.

Herrman C. et al (1996) J. Biol. Chem. 271(12): 6794-6800.

2.

Alberts et al (1983) Molecular Biology of the Cell, 3rd Edition: 206-207.

3.

Kontani K. et al (2002) J. Biol. Chem. 277(43): 41070-41078.

4.

Adeji A. et al (2001) J. National Cancer Institute 93(14): 1062-1074.

activemotif.com|

12Troubleshooting Guide
Problem/Question

Possible cause

Recommendation

No signal or weaksignal in all wells

Omission of key reagent

Check that all reagents have been added in all wells in
the correct order.

Substrate or conjugate is
no longer active

Test conjugate and substrate for activity by mixing HRP
and Chemiluminescent Working Solution together.

Enzyme inhibitor present

Sodium azide will inhibit the peroxidase reaction. Follow
our recommendations to prepare buffers.

Plate reader settings
not optimal

Verify the measurement mode and filter settings on theplate reader.

Incorrect
assay temperature

Bring substrate to room temperature before using.

Inadequate volumeof ChemiluminescentWorking Solution

Check to make sure that correct volume is deliveredby pipette.

Measurement time
too long

Reduce integration time or exposure time on
luminometer or CCD camera.

Concentration of
antibodies too highIncrease antibody dilutions.

Inadequate washing

Ensure all wells are filled with Wash Buffer and follow
washing recommendations.

Too much cellular extract
per wellDecrease amount of cellular extract.

Concentration of
antibodies is too high

Perform antibody titration to determine optimal working
concentration. Start by using 1:1,000 for primary antibody
and 1:10,000 for secondary antibody. The sensitivity of
the assay will decrease.

Not enough extract
per wellIncrease amount of extract. Do not exceed 500 µg/well.

Ras is poorly activatedor inactive.

Perform a time course for Ras activation in the studied
cell line.

Extracts are not fromcorrect species

Refer to cross-reactivity information on page 4.

High background in
all wells

High background insample wells

No signal or
weak signal insample wells

activemotif.com|

13

Technical Services

China

Japan

EuropeNorth America

If you need assistance at any time, please call or send an e-mail to Active Motif Technical
Service at one of the locations listed below.

Toll free:877 222 9543

Direct:

760 431 1263

Fax:

760 431 1351

E-mail:tech_service@activemotif.com

Direct:+32 (0)2 653 0001

Fax:

+32 (0)2 653 0050

E-mail:eurotech@activemotif.com

Direct:+81 (0)3 5225 3638

Fax:

+81 (0)3 5261 8733

E-mail:japantech@activemotif.com

Direct:(86)-21-20926090

Cell Phone:
E-mail:18521362870
techchina@activemotif.com

activemotif.com|

14