KRAS In-well Lysis ELISA Kit
Catalog No. 52100 & 52105(version A1)
Copyright 2023 Active Motif, Inc.

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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 in thismanual 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 UnitedStates and international treaties. The manufacturer of this documentation is Active Motif, Inc.
© 2023 Active Motif, Inc., 1914 Palomar Oaks Way, Suite 100; Carlsbad, CA 92008. All rights
reserved.
For Research Use Only. Not for use in diagnostic procedures. Permitted Use; Resale Prohibited
In the absence of an express written agreement to the contrary, all products are sold andservices deliverables are provided by Active Motif for (a) internal in vitro research purposes only
and may not be used for services or any other commercial purpose (b) the exclusive use of theoriginal purchaser, and are not to be resold. You agree not to reverse engineer or otherwise
attempt to discover the structure or composition of products or services deliverables unless weotherwise agree in writing.

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TABLE OF CONTENTS

Page

Overview  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Flow Chart of Process . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Kit Performance and Benefits  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Kit Components and Storage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
Additional Materials Required  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Protocols
Buffer Preparation and Recommendations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
Quick Chart for Preparing Buffers  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
In-well Cell Lysis and ELISA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Appendix
Section A. Preparation of Cell Extract  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
Section B. Troubleshooting Guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Technical Services . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

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Overview
Small GTPase RAS proteins bind to and hydrolyze GTP, allowing them to function as molecularswitches, cycling between an active (GTP-bound) and inactive (GDP-bound) state. This switch
regulates signal transduction pathways involved in cellular functions including proliferation,differentiation, and apoptosis. RAS proteins exist as three major isoforms: HRAS, NRAS, andKRAS, each containing about 190 amino acids that share 80-90% sequence identity.
RAS is one of the most frequently mutated genes in cancer. Gain-of-function missense mutations
which promote oncogenesis cluster at codons 12, 13, and 61 of KRAS, NRAS and HRAS resulting
in enhanced GTP binding due to fast exchange of nucleotide and/or impairment of GAP(GTPase Activating Protein) binding. Although RAS mutations are all activating, they vary in their
oncogenic potential and frequency in different tissues, with KRAS mutations being the mostprevalent.
The KRAS In-Well ELISA Kit, which measures active human KRAS via chemiluminescent detection,is an extension of the existing RAS GTPase Chemi ELISA Kit (Cat no. 52097). The KRAS In-Well
ELISA Kit is designed specifically for the study of KRAS activation and inhibition. The new assay
allows for direct in-well lysis of cells cultured in 96-well plate format, either directly in media or
following a PBS wash step. The KRAS In-well ELISA Kit contains a Raf-RBD protein fused to GST
that will be coated onto the provided 96-well, glutathione-coated plate. Active RAS containedin the cellular extract binds to Raf-RBD, while inactive RAS does not and is washed away. Bound
KRAS is then detected by incubation with a primary antibody that specifically detects KRAS.
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.

product

formatcatalog no.

KRAS In-well Lysis ELISA Kit

1 x 96 rxns

52100

KRAS In-well Lysis ELISA Kit

5 x 96 rxns

52105

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1Flow Chart of Process

21

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01

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2

1. Bind GST-Raf-RBD to ELISA plate

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A
B

C
D
E
F

1 hr. (4°C)

G
H

2. Add 5X Complete Lysis Binding
Buffer to cell culture plate

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15 min. (on ice)
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3. Transfer cell lysate from cell
culture plate to ELISA

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1 hr. (RT)
4. Add KRAS antibody

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1 hr. (RT)
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5. Add anti-IgG HRP antibody

A
B
C
D
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1 hr. (RT)

6. Add ChemiluminescentWorking Solution

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Kit Performance and Benefits
The KRAS In-well ELISA Kit is for research use only. Not for use in diagnostic procedures.
Recommended cell seeding density: The optimal cell seeding density for a 96 well culture plate
for use in the ELISA will depend on both the cell line and incubation time. Guidelines are given
below for two cell lines.
HeLa: 7.0 x 104 - 16 x 104 cell seeding density
Aspc1: 2.0 x 104 - 32 x 104 cell seeding density
Range of Detection: The KRAS In-well Lysis ELISA kit will give relative quantitation of KRAS in therange of 6.25 µg - 50 µg whole cell extract/well.
Cross-reactivity: The KRAS In-well Lysis ELISA kit is human KRAS specific. It does not detect thehuman HRAS or NRAS isoforms.
Assay time: 4.5 hours.

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3Kit Components and Storage
Please store each component at the temperature indicated in the table below upon receipt of thekit. Kit components must be stored at the temperatures listed below for 24 hours prior to use.

Catalog no.

52100

52105

Reactions96

5 x 96

GST-Raf-RBD

110 µl5 x 110 µl-80°C

Positive Control Lysate (AsPC-1)

42 µl

5 x 42 µl-80°C

Recombinant KRAS Antibody

15 µl

44 µl

-20°C

Anti-rabbit HRP-conjugated IgG

15 µl

15 µl

-20°C

Protease Inhibitor Cocktail (PIC)

2 x 105 µl

1.2 ml-20°C

5X Lysis Binding Buffer

4 ml

22 ml

4°C

10X Wash Buffer AM2

2 x 25 ml

2 x 125 ml

4°C

2.5 ml12.5 ml

4°C

Chemiluminescent Reagent

2 ml

10 ml

4°C

Reaction Buffer

4 ml

20 ml

4°C

96-well assay plate

1 ea

5 ea

4°C

Plate sealer

2 ea

10 ea

4°C

10X Antibody Binding Buffer AM2

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StorageAdditional materials required• 96 well microplates• 5 or 10 ml Falcon™ tubes• 2-200 µl multi-channel pipettes• Reagent Reservoirs• Pipettes• Pipette tips
• 1.5 ml microtubes• Microplate shaker• Vortex• Rocking platform at room temperature and 4°C• Distilled water• HandyStep Repeating Pipette with syringe tips (optional)• Automated plate washer (optional)• 1X PBS (Optional for washing cells) pH 7.4-7.6• Microplate luminometer or CCD camera-coupled imaging system

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5

Protocols
Buffer Preparation and Recommendations
Preparation of 5X Complete Lysis Binding Buffer(For In-well Lysis in cell culture plate)
Preparation of 5X Complete Lysis Binding Buffer is required for in-well lysis without cell wash. For
a 96 well culture plate, prepare a total of 3.0 ml 5X Complete Lysis Binding Buffer by mixing 2.85
ml 5X Lysis Binding Buffer with 150 µl Protease Inhibitor Cocktail. Protease inhibitors lose theiractivity after 24 hours once diluted, so make the 5X Complete Lysis Binding Buffer the day of use.
The remaining amount should be discarded if not used in the same day.
Preparation of 1X Complete Lysis Binding Buffer(For Binding GST-Raf-RBD to ELISA plate)
Preparation of 1X Complete Lysis Binding Buffer is required for binding the RBD-Raf-RBD protein
to the ELISA Plate. For a 96 well ELISA, prepare 5.0 ml 1X Complete Lysis Binding Buffer by
mixing 50 µl Protease Inhibitor Cocktail, 1.0 ml 5X Lysis Binding Buffer, and 3.95 ml distilledwater. Protease inhibitors lose their activity after 24 hours once diluted, so make the 1X Complete
Lysis Binding Buffer the day of use. The remaining amount should be discarded if not used in thesame day.
GST-Raf-RBD
The GST-Raf-RBD contains a RAS Binding Domain and is used to capture activated RAS on theglutathione-coated plate. Each vial of GST-Raf-RBD contains enough to coat one 96 well ELISA
plate. If only a partial plate will be used, aliquot into small fractions to avoid freeze/thaws andstore at -80°C. For a 96 well plate prepare 5.0 ml GST-Raf-RBD Solution by adding 100 µl GSTRaf-RBD to 4.9 ml 1X Complete Lysis Binding Buffer. Invert 10 times to mix. Discard any unused
diluted GST-Raf-RBD.
Preparation of 1X Wash Buffer
For each 96 well ELISA prepare 300 ml of 1X Wash Buffer by mixing 30 ml of 10X Wash Buffer
with 270 ml distilled water. 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. If this occurs,
homogenize the buffer by mixing gently.
Preparation of 1X Antibody Binding Buffer
For each 96 well ELISA prepare 13 ml 1X Antibody Binding Buffer by mixing 1.3 ml of 10X Antibody
Binding Buffer with 11.7 ml distilled water. Mix gently to avoid foaming. Discard any remaining 1X
Antibody Binding Buffer after use. If the BSA contained in the 10X Antibody Binding Buffer forms
clumps, homogenize the buffer by warming to room temperature and inverting gently several
times.
Diluted KRAS Antibody
The primary KRAS antibody recognizes human KRAS. Spin vial briefly in a microcentrifuge before
opening to collect contents to the vial bottom. The supplied antibody will be diluted 1:1,000 in 1X
Antibody Binding Buffer. For a 96 well plate, prepare 6.0 ml of Diluted KRAS Antibody by mixing
6.0 µl Recombinant KRAS Antibody with 6 ml 1X Antibody Binding Buffer. Invert 10 times to mix.
If not using the entire vial of Recombinant KRAS antibody at one time we suggest aliquoting into
smaller volumes to avoid multiple freeze and thaw cycles.
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Diluted Anti-rabbit HRP-conjugated IgG
The Anti-rabbit HRP-conjugated IgG is used as the secondary antibody to detect bound primary
antibody. Spin vial briefly in a microcentrifuge before opening to collect contents to the vial
bottom. The supplied antibody will be diluted 1:10,000 in 1X Antibody Binding Buffer. This dilution
should be made by first performing a 1:10 dilution followed by a 1:1000 dilution. For a 96 wellplate, first combine 2 µl anti-rabbit HRP-conjugated IgG with 18 µl 1X Antibody Binding Buffer. Mixby pipetting up and down. Then add 6.0 µl of this mixture to 6 ml 1X Ab Binding Buffer. Invert 10
times to mix. If not using the entire vial of Anti-rabbit HRP-conjugated IgG at one time we suggest
aliquoting into smaller volumes to avoid multiple freeze and thaw cycles.
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 andReaction Buffer at room temperature for at least 1 hour. These reagents are mixed in a 1:2 ratio tocreate the Chemiluminescent Working Solution. For a 96 well plate, mix 1.9 ml Chemiluminescent
Reagent with 3.8 ml volumes of Reaction Buffer. Invert several times to mix. The Chemiluminescent Working Solution is stable for several hours. After use, discard any remaining Chemiluminescent Working Solution.
Positive Control Lysate (AsPC-1)
The Positive Control Lysate (AsPC-1 ) is provided as a positive control for KRAS activation. Eachvial contains enough material for 4 reactions per plate. The extract is optimized to give a strong
signal with an assay window (signal/background) of more than 10X when used at 10 μl/well. For
best results we recommend thawing it no more than 15 minutes prior to use. Avoid freeze/thaw
cycles of the extract and store at -80ºC.

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7Quick Chart for Preparing Buffers**** The Quick Chart includes an excess of components to perform the assay.
Quick Chart (Per 96 well plate)
Reagents

Components

Volume5X Complete Lysis Binding Buffer(For In-well Cell Lysis)

Protease Inhibitor Cocktail
5X Lysis Binding Buffer
Total

150 µl2.85 ml
3.00 ml

1X Complete Lysis Binding Buffer (For ELISA Assay)

Protease Inhibitor Cocktail
5X Lysis Binding Buffer
Distilled water
Total

50 µl
1.0 ml3.95 ml
5.0 ml

GST-Raf-RBD for coating plate

GST-Raf-RBD
1X Complete Lysis Binding
Total

100 µl
4.9 ml
5.0 ml

10X Wash Buffer
Distilled water
Total

30 ml
270 ml300 ml1X Antibody Binding Buffer

10X Antibody Binding Buffer
Distilled waterTotal

1.3 ml11.7 ml
13 ml

Diluted KRAS Antibody (1/1000 dilution)

Recombinant KRAS Antibody
1X Antibody Binding Buffer
Total

6.0 µl
6.0 ml
6.0 mlAnti-rabbit HRP-conjugated IgGPre-Dilution

Anti-rabbit HRP-conjugated IgG
1X Antibody Binding Buffer
Total

2 µl
18 µl
20 µl

Diluted Anti-rabbit HRP-conjugated IgG

Anti-rabbit HRP- conjugated IgG
Antibody Pre-Dilution
1X Ab Binding Buffer
Total

6.0 µl
6.0 ml
6.0 mlChemiluminescent Reagent
Reaction Buffer
Total

1.9 ml3.8 ml5.7 ml

1X Wash Buffer (can store for 1 week, 4C)

Chemiluminescent Working Solutionwww.activemotif.com

8

Protocol
PLEASE READ ENTIRE PROTOCOL BEFORE STARTING
For optimal kit performance, kit components must be stored at the recommended storage
temperatures indicated on page 4 of the manual for 24 hours prior to use. Prepare the 5X
Complete Lysis Binding Buffer, 1X Complete Lysis Binding, 1X Wash Buffer and 1X Antibody
Binding Buffer as described in the section Buffer Preparation and Recommendations. A multichannel pipette or HandyStep Repeating pipette and reagent reservoirs may be used fordispensing all reagents.
The protocol below is for the in-well lysis workflow. Alternatively, whole cell extracts can beprepared separately as described in the Appendix, Section A. Preparation of Whole-Cell Extract,
diluted in 1X Complete Lysis Binding Buffer and transferred to the ELISA.
Step 1: Treatment of Cells
The number of seeded cells and incubation period should be optimized for each cell line. It is
recommended that a range of 5 x 103 cells to 1.6 x 106 cells be used per well. After seeding cells,
an incubation of at least 2 days is recommended to stabilize cultured cells before treatment. We
suggest 3-4 replicates per experimental condition.
Treat the cells with 100 µl of media containing KRAS activating or inhibiting components as
required.
For best results, the GST-Raf-RBD should be freshly bound to the ELISA plate immediately before
cell lysis. We suggest doing Step 2: Binding the GST-Raf-RBD to Plate, 1 hour before KRAS activation/inhibition will be complete. Then proceed with Step 3: In-well Cell Lysis.
Step 2: Binding GST-Raf-RBD to Plate
1.

Thaw the GST-Raf-RBD on ice immediately before use. For a full plate mix 100 µl GST-RafRBD with 4.9 ml 1X Complete Lysis Binding Buffer. Invert 10 times to mix. Dispense 50 µl
GST-Raf-RBD mixture into each 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 plate over awaste container to empty the wells, then tap the inverted plate 3 times on absorbent paper
towels. Alternatively, a microplate washer may be used.

Step 3: In-well Lysis Cell Lysis4.

Add 25 µl 5X Complete Lysis Binding Buffer to each well of the culture plate using a
multichannel pipettor. Mix by pipetting up and down 3-5 times.
NOTE: Alternatively, culture media can be removed, and wells washed with 200 µl PBS,
before adding 125 µl 1X Complete Lysis Binding Buffer to each well.

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5.

Firmly seal the plate. Place the sealed plate on ice for 15 minutes. For some cell lines, lysis
may be improved by placing the plate on a microplate shaker at 100-120 rpm and 4°C for 15
minutes.

6.

Carefully remove the plate seal. For the assay, 50 µl of lysed whole cells from each well
will be transferred to the ELISA plate. Be sure the lysed cells are resuspended before thesample is transferred. Cell lysates can be stored frozen at -80 C before using in the ELISA.
However, a reduction in the ELISA signal may be observed.

Step 4: Add Samples to Wells
Sample wells: Transfer 50 µl of lysed whole cells from each culture well to the ELISA for theassay. Be sure the lysed cells are resuspended by pipetting up and down 3-5 times before thesample is transferred.
Positive control wells: Thaw the Positive Control Lysate (AsPC-1) on ice for no more than 15
minutes prior to use. Mix 10 µl of extract with 40 µl of 1X Complete Lysis/Binding Buffer per well.
Transfer 50 µl to each positive control well.
Blank wells: Add 50 µl 1X Complete Lysis/Binding Buffer only per well.
7.

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

8.

Wash each well 3 times with 200 µl 1X Wash Buffer. For each wash, flick the plate over awaste container to empty the wells, then tap the inverted plate 3 times on absorbent paper
towels. Alternatively, a microplate washer may be used.

Step 5: Binding of primary antibody
9.

Add 50 µl diluted KRAS antibody (1:1000 dilution in 1X Antibody Binding Buffer) to wells.

10. Cover the plate and incubate for 1 hour at room temperature without agitation.
11.

Wash each well 3 times with 200 µl 1X Wash Buffer. For each wash, flick the plate over awaste container to empty the wells, then tap the inverted plate 3 times on absorbent paper
towels. Alternatively, a microplate washer may be used.

Step 6: Binding of secondary antibody
12. Add 50 µl diluted HRP antibody (1:10,000 dilution in 1X Antibody Binding Buffer) to all wells
being used.
13. Cover the plate and incubate for 1 hour at room temperature without agitation.
14. During this incubation, place Chemiluminescent Reagent and Reaction Buffer at room
temperature.
15. Wash each well 3 times with 200 µl 1X Wash Buffer. For each wash, flick the plate over awaste container to empty the wells, then tap the inverted plate 3 times on absorbent paper
towels. Alternatively, a microplate washer may be used.

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10Step 7: Chemiluminescent detection
16. Add 50 µl room-temperature Chemiluminescent Working Solution to all wells being used.
Read chemiluminescence using a luminometer. Readings should be taken within 15 minutes
to minimize changes in signal intensity. For experiments involving multiple plates, it isrecommended to stagger the addition of the chemiluminescent solution so that each plateis measured within the same time frame

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Appendix
Section A. Preparation of Whole-Cell Extract
To prepare whole-cell extracts (without in-well lysis) for use in the ELISA, the following protocol
can be used for a 100 mm culture dish or 2 x 107 cells. Additional 5X Lysis Binding Buffer andProtease Inhibitor Cocktail can be purchased as Cat. no. 52110.
1.

For each 100 mm culture dish or 2 x 107 cells, prepare 500 μl 1X Complete Lysis Binding
Buffer by mixing 5 µl Protease Inhibitor Cocktail, 100 μl 5X Lysis Binding Buffer, and 395 μl
distilled water.
NOTE: Protease inhibitors lose their activity after 24 hours once diluted, so make the 1X
Complete Lysis Binding Buffer the day of use. The remaining amount should bediscarded if not used the same day.

2.

Remove culture media from cells and wash with 5 ml ice-cold PBS.

3.

For adherent cells add 500 μl of 1X Complete Lysis Binding Buffer to the plate and scrape
cells with a rubber policeman. For pelleted cells resuspend cell pellet in 500 μl 1X Complete
Lysis Binding Buffer.

4.

Transfer suspended cells to a microcentrifuge tube. Incubate 15 minutes on ice.

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.

7.

Measure the protein content using a Bradford-based assay.

8.

Extracts can be aliquoted and frozen at -80°C for later use. However, for best results, use
immediately in the ELISA. Dilute whole-cell extracts in 1X Complete Lysis Binding Buffer todesired concentration and transfer 50 μl to each ELISA well.

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12Section B. Troubleshooting Guide
Problem/Question

Possible cause

Recommendation

No signal or weak signal 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.

Plate reader settings not
optimalVerify that measurement mode is set toluminescence.

Incorrect assay
temperature

Bring substrate to room temperature before
using.

Inadequate volumeof ChemiluminescentWorking Solution

Check to make sure that correct volume isdelivered by 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.

High background in sample
wells

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. Check calibration of
pipettes. Background in this assay directly
correlates with antibody concentration.

No signal or weak signal insample wells

Not enough extract per
well

Increase number of cells seeded in theculture plate.

KRAS is poorly activatedor inactive

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

Extracts are not fromcorrect species

Refer to cross-reactivity information on page3.

High background in all wells

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13

Technical Services
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.

Active Motif North America
Toll free: 877.222.9543
Direct: 760.431.1263
Fax: 760.431.1351
E-mail: tech_service@activemotif.com
Active Motif Europe
UK Free Phone:France Free Phone:
Germany Free Phone:Direct:Fax:
E-mail:0800/169 31 47
0800/90 99 79
0800/181 99 10+32 (0)2 653 0001
+32 (0)2 653 0050
eurotech@activemotif.com

Active Motif Japan
Direct: +81 (0)3 5225 3638
Fax: +81 (0)3 5261 8733
E-mail: japantech@activemotif.com
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Direct: (86)-21-20926090
Cell Phone: 18521362870
E-mail: techchina@activemotif.com

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NotesNotes