TransAM™

NFATc1 Transcription Factor
Assay Kits
(version D2)
Catalog Nos. 40296 & 40796

TABLE OF CONTENTS

Page

Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Flow Chart of Process . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Introduction
NFATc1 Transcription Factor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Transcription Factor Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
TransAM NFATc1. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
Kit Performance and Benefits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Kit Components and Storage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
Protocols
Buffer Preparation and Recommendations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
Quick Chart for Preparing Buffers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
NFATc1 Transcription Factor Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Preparation of Nuclear Extract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
Appendix
Section A. Troubleshooting Guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Technical Services . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

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Overview
The transcription factor known as Nuclear Factor of Activated T cells (NFAT) binds to several
sites within the regulatory region of the IL-2 gene and other genes induced during the immune
response. Because its activation is inhibited by the main immunosuppressive drugs in current
clinical use, cyclosporin A and tacrolimus (FK506), accurate monitoring of NFAT activation incells, tissues and animals is crucial for biomedical research and drug development. To date, suchresearch projects are tedious and time consuming, and lack high-throughput screening methods.
With its patented TransAM™ method*, Active Motif introduced the first ELISA-based kits to detectand quantify transcription factor activation. TransAM Kits combine a fast, user-friendly format
with a sensitive, specific assay. TransAM NFAT Kits are designed specifically for the study of the
NFAT activation pathway. They contain a 96-well plate to which an oligonucleotide containing
the NFAT consensus binding site has been immobilized. NFAT contained in nuclear extracts binds
specifically to this oligonucleotide and is detected through use of an antibody directed against
NFATc1. Addition of a secondary antibody conjugated to horseradish peroxidase (HRP) provides
sensitive colorimetric readout that is easily quantified by spectrophotometry. The 96-well platewith individual strips of 8 wells is suitable for manual use or for high-throughput screening applications. TransAM NFATc1 Kits are available in two sizes:
product
TransAM NFATc1

formatcatalog no.

1 x 96-well plate5 x 96 well plates

40296
40796

See Active Motif products related to the NFAT transcription factor in Appendix, Section B.

* Technology covered by AAT-filed patents and licensed to Active Motif.

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1Flow Chart of Process

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2

Introduction
NFAT Transcription Factors
NFAT proteins are transcription factors that were first identified as inducers of the immune
response1. As demonstrated later, these proteins also play varied roles in cell differentiation andadaptation for vascular endothelial cells or skeletal muscle cells (see 2 for review). There are
four NFAT family members (NFAT1-4, NFAT2 is also called NFATc or NFATc1) from which numerous
isoforms are generated by alternative splicing1. NFAT mRNAs are found in peripheral blood lymphocytes, spleen (NFAT1 and 2) and thymus (NFAT4). NFATs modulate the expression of numerous cytokines such as IL-2, IL-3, IL-4, IL-5, IL-8, IL-13, GM-CSF, IFNα, IFNγ and CD40L1. In resting
cells, NFATc1 is confined to the cytoplasm, where it is maintained in a phosphorylated state by
the action of constitutive kinases. Upon stimulation, NFATc1 is dephosphorylated by calcineurin,a Ca2+-dependent phosphatase (see 3 for review), and migrates to the nucleus. NFATc1 dephosphorylation is stimulated by Ca2+-coupled membrane receptors, such as T cell and B cell receptors, and the CD40, FceRI, CD16 and G protein-associated receptors (thrombin or H1 histamine
receptors). NFATc1 can also be activated by calcium ionophores. Cyclosporin A and FK506 immunosuppressor drugs inhibit calcineurin activity on NFAT. Receptors not associated with calcium
movement are not expected to stimulate NFAT1. When calcium levels drop, calcineurin becomesinactive, and NFATc1 is rephosphorylated by kinases and exported back into the cytoplasm.
NFAT phosphoproteins share two conserved domains: a DNA-binding domain (DBD) displaying
limited similarity to the Rel protein family DBD4, and modulating interactions with AP-1 dimers;
and a NFAT homology region (NHR), upstream of the DBD, that regulates translocation and DNAbinding activity. The regulatory domain is dephosphorylated by the calcium- and calmodulindependent phosphatase calcineurin, which controls NFAT nuclear translocation. Transactivation
domains can be found at the N- and C-terminal ends of the NFAT proteins5. NFATs bind to the
DNA consensus motif 5´-T/AGGAAA-3´ as monomers6. NFATs can cooperatively interact with AP-1
and GATA proteins for DNA binding7, 8. NFAT can also bind to certain kB-like sites9. Vitamin D3
receptor heterodimers (RXR:VDR) can abrogate NFAT modulation of IL-2 by binding to a sitewhich overlaps the NFAT distal site10. Sites in IL-2 and GM-CSF promoters can accommodate
both NFAT and the Ets-family member, Elf-111, 12.

Transcription Factor Assays
To date, three methods are widely used to measure NFAT activation, either directly or indirectly:
1.

NFAT activation can be determined by Western Blot by using antibodies specific for NFAT
proteins. This method is time consuming (up to 2 days once the nuclear extracts are prepared) and is not suitable for processing large numbers of samples.

2.

The DNA-binding capacity of NFAT can be assayed by gel retardation, also called electrophoretic mobility shift assay (EMSA). In this method, nuclear extracts are incubated witha radioactive double-stranded oligonucleotide probe containing the consensus sequence
for NFAT binding. If NFATs are active in the nuclear extract, they will bind to the probe.
Samples are then resolved by electrophoresis on a native polyacrylamide gel, followedby autoradiography. This method is sensitive, but like the previous procedure, it is time
consuming (multiple days of gel exposure may be required to achieve sufficient sensitivity)
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3and it cannot be applied to high-throughput screening. Gelshift assays also require special
precautions and equipment for handling radioactivity.
3.

Another method used to assay NFAT activation is based on reporter genes, typically luciferase or β-galactosidase, placed under the control of a promoter containing a NFAT consensus binding site. The promoter can be artificial, made of several NFAT sites and a TATA box,
or natural, like promoter sequences from cytokine genes. However, the procedure is limited by the following issues: (i) reporter gene assays have to be repeated several times toobtain statistically reliable data; and (ii) reporter gene assays are sensitive to confounding
factors that may influence the expression level of the reporter gene, and therefore assays
have to be carefully standardized. Reporter gene assays are sensitive and easy to performwith a large number of samples, but require efficient cell transfection with the reporter
plasmid.

TransAM NFATc1
NFAT-regulated genes are mainly involved in immune response. Recent studies have shown that
NFATs also regulate cell development and cellular adaptation. However, this field has been hampered by the lack of convenient assays suitable for discriminating the NFAT family members andfor performing high sample number experiments.
To overcome this, Active Motif is introducing a high-throughput assay to quantify NFATc1
activation13. The TransAM Kit combines a fast and user-friendly ELISA format with a sensitive and specific assay for transcription factors. TransAM NFAT Kits contain a 96-well plate on
which has been immobilized an oligonucleotide that contains a NFAT consensus binding site
(5´-AGGAAA-3´). NFATs contained in nuclear extract specifically bind to this oligonucleotide.
The primary antibody used in TransAM NFATc1 Kit recognizes an accessible epitope on NFATc1
protein upon DNA binding. Addition of a secondary HRP-conjugated antibody provides a sensitive colorimetric readout easily quantified by spectrophotometry. Once the nuclear extracts areprepared, this assay is completed in less than 3.5 hours. As this assay is performed in 96-wellplates, a large number of samples can be handled simultaneously, enabling high-throughput
automation. This assay is specific for NFATc1 activation and has been shown to be 10-fold moresensitive and 20-fold faster than the gel-retardation technique. With the 3.5-hour TransAM procedure, we could detect NFATc1 activation using as little as 0.6 µg of nuclear extract. A comparable assay using EMSA required 5 µg of nuclear extract and a 2-day autoradiography.
TransAM NFATc1 has many applications including the study of NFATc1-mediated transcriptional
regulation, protein structure/function studies of NFATc1, NFATc1 cytoplasmic/nuclear shuttling and
NFATc1 antagonist drug validation in areas such as immune response, angiogenesis, myogenesis, and many more.

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4Kit Performance and Benefits
The TransAM NFATc1 Kit is for research use only. Not for use in diagnostic procedures.
Detection limit: < 0.6 µg nuclear extract/well. TransAM NFATc1 is 10-fold more sensitive than
EMSA.
Range of detection: TransAM provides quantitative results from 0.6 to 10 µg of nuclear extract/well (see graph below).
Cross-reactivity: TransAM NFATc1 detects NFATc1 from human and mouse origin.

NFATc1 activation (OD450 nm)

Assay time: 3.5 hours. TransAM is 20-fold faster than EMSA.

1.25

JurkatJurkat + PHA

1
0.75
0.5

0.25
0

0.625

10
1.25
2.5
5
Nuclear extract (µg/well)

Monitoring NFATc1 activation with the TransAM NFATc1 Kit: Different amounts of nuclear extract from
untreated and PHA treated Jurkat cells are tested for NFATc1 activation by using the TransAM NFATc1 Kit. Thisdata is provided for demonstration only.

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5Kit Components and Storage
Except for the nuclear extract that must be kept at -80ºC, kit components can be stored at -20ºCprior to first use. Then, we recommend storing each component at the temperature indicated in
the table below.
Reagents

Quantity
1 plate / 5 plates

Storage / Stability

NFATc1 antibody

22 µl / 110 µl(0.2 mg/ml)

4°C for 6 months

Anti-mouse HRP-conjugated IgG

11 µl / 55 µl(0.4 mg/ml)

4°C for 6 months

Wild-type oligonucleotide AM9100 µl / 500 µl(10 pmol/µl)-20°C for 6 months

Mutated oligonucleotide AM9100 µl / 500 µl(10 pmol/µl)-20°C for 6 months

Jurkat nuclear extract (PHA treated)

40 µl / 200 µl(2.5 mg/ml)-80°C for 6 months

Dithiothreitol (DTT)

100 µl / 500 µl(1 M)-20°C for 6 months

Protease Inhibitor Cocktail

100 µl / 500 µl-20°C for 6 months

Lysis Buffer AM1

10 ml / 50 ml

4°C for 6 months

Binding Buffer AM1

10 ml / 50 ml

4°C for 6 months

10X Wash Buffer AM2

22 ml / 110 ml

4°C for 6 months

10X Antibody Binding Buffer AM3

2.2 ml / 11 ml

4°C for 6 months

Developing Solution

11 ml / 55 ml

4°C for 6 months

Stop Solution

11 ml / 55 ml

4°C for 6 months

96-well NFAT assay plate

1/5

4°C for 6 months

Plate sealer

1/5

Additional materials required

For Nuclear Extract preparation• Multi-channel pipettor• Hypotonic Buffer• Multi-channel pipettor reservoirs• Rocking platform• Phosphatase Inhibitor Buffer• Microplate spectrophotometer capableof reading at 450 nm (655 nm as
optional reference wavelength)• Detergent (NP-40)

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6

Protocols
Buffer Preparation and Recommendations
Preparation of Complete Lysis Buffer
We provide an excess of Lysis Buffer AM1 in order to perform the assay AND to prepare customized cell extracts. Our Nuclear Extract Kit can also be purchased separately (Cat. Nos. 40010 &
40410). Prepare the amount of Complete Lysis Buffer required for the assay by adding 1 µl of 1
M DTT and 10 µl of Protease Inhibitor Cocktail per ml of Lysis Buffer AM1 (see the Quick Chartfor Preparing Buffers in this section). Some of the protease inhibitors lose their activity after 24hours once diluted. Therefore, we recommend using the Complete Lysis Buffer immediately forcell lysis. The remaining amount should be discarded if not used in the same day.
Binding BufferThis is supplied ready to use.
Preparation of 1X Wash Buffer
Prepare the amount of 1X Wash Buffer required for the assay as follows: For every 100 ml of
1X Wash Buffer required, dilute 10 ml 10X Wash Buffer AM2 with 90 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 the 10X Wash Buffer AM2
may form clumps, therefore homogenize the buffer by incubating at 50ºC for 2 minutes and mixing 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 AM3 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 AM3 may form clumps, therefore homogenize the buffer by warming toroom temperature and vortexing for 1 minute prior to use. Dilute primary and HRPconjugated secondary antibodies to 1:500 and 1:1000, respectively, with the 1X Antibody Binding
Buffer. Depending on the particular assay, the signal:noise ratio may be optimized by using
higher dilutions of both antibodies. This may decrease the sensitivity of the assay.
* Volumes listed refer to the preparation of buffer for diluting both the primary & secondary antibodies.

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7Developing Solution
The Developing Solution should be warmed to room temperature before use. The Developing
Solution is light sensitive, therefore, we recommend avoiding direct exposure to intense lightduring storage. The Developing Solution may develop a yellow hue over time. This does notaffect product performance. A blue color present in the Developing Solution indicates that it
has been contaminated and must be discarded. Prior to use, place the Developing Solution atroom temperature for at least 1 hour. Transfer the amount of Developing Solution required forthe assay into a secondary container before aliquoting into the wells (see the Quick Chart for
Preparing Buffers in this section). After use, discard remaining Developing Solution.
Stop Solution
Prior to use, transfer the amount of Stop Solution required for the assay into a secondary
container (see the Quick Chart for Preparing Buffers in this section). After use, discard remaining
Stop Solution.
WARNING:

The Stop Solution is corrosive. Wear personal protective equipment whenhandling, i.e. safety glasses, gloves and labcoat.

Nuclear Extract
The Jurkat nuclear extract (PHA treated) is provided as a positive control for NFAT activation.
Sufficient extract is supplied for 20 reactions per plate. This extract is optimized to give a strong
signal when used at 5 µg/well. We recommend aliquoting the extract in 5 µl fractions and storingat -80ºC. Avoid multiple freeze/thaw cycles of the extract. Various cell extracts are available from
Active Motif (see Appendix, Section B. Related Products).
Wild-type and mutated consensus oligonucleotides
The wild-type consensus oligonucleotide is provided as a competitor for NFAT binding in orderto monitor the specificity of the assay. Used at 20 pmol/well, the oligonucleotide will prevent
NFAT binding to the probe immobilized on the plate. Conversely, the mutated consensus oligonucleotide should have no effect on NFAT binding. Prepare the required amount of wild-type
and/or mutated consensus oligonucleotide by adding 2 µl of appropriate oligonucleotide to 43µl of Binding Buffer AM1 per well being used (see the Quick Chart for Preparing Buffers in thissection). To allow for optimum competition, add the oligonucleotide to the well prior to additionof the cell extract.

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8Quick Chart for Preparing Buffers
Reagents to prepare

Components

For 1
well

Complete Lysis Buffer

DTT
Protease inhibitor cocktail
Lysis Buffer AM1

0.01 µl0.12 µl11.12 µl

For 1 strip For 6 strips For 12 strips(8 wells) (48 wells) (96 wells)
0.1 µl0.9 µl89 µl

0.6 µl5.4 µl534 µl1.2 µl10.8 µl
1.068 ml

TOTAL REQUIRED

11.25 µl

90 µl

540 µl1.08 ml

Binding Buffer AM1

TOTAL REQUIRED

45 µl

360 µl2.16 ml

4.32 ml

Binding Buffer withwild-type ormutated oligonucleotide

Wild-type or mutated oligo
Binding Buffer AM1

TOTAL REQUIRED

2 µl
43 µl

45 µl

16 µl
344 µl360 µl

96 µl
2.064 ml

2.16 ml

N/A
N/A

N/A

1X Wash Buffer

Distilled water
10X Wash Buffer AM2

TOTAL REQUIRED

2.025 ml
225 µl2.25 ml

16.2 ml
1.8 ml18 ml

97.2 ml
10.8 ml

108 ml194.4 ml21.6 ml

1X Antibody
Binding Buffer*

Distilled water
10X Ab Binding Buffer AM3

202.5 µl22.5 µl

1.62 ml
180 µl

1.8 ml9.72 ml
1.08 ml

10.8 ml19.44 ml
2.16 ml21.6 ml

Developing Solution

TOTAL REQUIRED

112.5 µl

900 µl5.4 ml10.8 ml

Stop Solution

TOTAL REQUIRED

112.5 µl

900 µl5.4 ml10.8 ml

TOTAL REQUIRED

225 µl216 ml* Volumes listed refer to the preparation of buffer for diluting both the primary & secondary antibodies.

NFATc1 Transcription Factor Assay
Determine the appropriate number of microwell strips required for testing samples, controls andblanks in duplicate. If less than 8 wells in a strip need to be used, cover the unused wells witha portion of the plate sealer while you perform the assay. The content of these wells is stable atroom temperature if kept dry and, therefore, can be used later for a separate assay. Store theunused strips in the aluminum pouch at 4°C. Use the strip holder for the assay.
Prepare the Complete Lysis Buffer, 1X Wash Buffer and 1X Antibody Binding Buffer as describedabove in the section Buffer Preparation and Recommendations. Multi-channel pipettor reservoirs
may be used for dispensing the Binding Buffer, Wash Buffer, Antibody Binding Buffer, Developing
Solution and Stop Solution into the wells being used.

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9

Step 1: Binding of NFAT to its consensus sequence1.

Add 40 µl Binding Buffer AM1 to each well to be used. If you wish to perform competitive
binding experiments, add 40 µl Binding Buffer AM1 that contains 20 pmol (2 µl) of the wildtype or mutated oligonucleotide (see the Buffer Preparation section above for a descriptionof competitive binding).

2.

Sample wells: Add 10 µl of sample diluted in Complete Lysis Buffer per well. We recommend using 2-10 µg of nuclear extract diluted in Complete Lysis Buffer per well. A protocol
for preparing nuclear extracts can be found on page 11.
Positive control wells: Add 5 µg of the provided nuclear extract diluted in 10 µl of Complete
Lysis Buffer per well (2 µl of nuclear extract in 8 µl of Complete Lysis Buffer per well).
Blank wells: Add 10 µl Complete Lysis Buffer only per well.

3.

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

4.

Wash each well 3 times with 200 µl 1X Wash Buffer. For each wash, flick the plate over asink to empty the wells, then tap the inverted plate 3 times on absorbent paper towels.

Step 2: Binding of primary antibody1.

Add 100 µl diluted NFATc1 antibody (1:500 dilution in 1X Antibody Binding Buffer) to all wells being
used.

2.

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

3.

Wash the wells 3 times with 200 µl 1X Wash Buffer (as described in Step 1, No. 4).

Step 3: Binding of secondary antibody1.

Add 100 µl of diluted anti-mouse HRP-conjugated antibody (1:1000 dilution in 1X Antibody
Binding Buffer) to all wells being used.

2.

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

3.

During this incubation, place the Developing Solution at room temperature.

4.

Wash the wells 4 times with 200 µl 1X Wash Buffer (as described in Step 1, No. 4).

Step 4: Colorimetric reaction
1.

Transfer the amount of Developing Solution required for the assay into a secondary container. Add 100 µl Developing Solution to all wells being used.

2.

Incubate 5-10 minutes at room temperature protected from direct light. Please read theCertificate of Analysis supplied with this kit for the optimal development time for this specific kit lot, which varies from lot to lot. Monitor the blue color development in the sample
and positive control wells until it turns medium to dark blue. Do not overdevelop.

3.

Add 100 µl Stop Solution. In presence of the acid, the blue color turns yellow.

4.

Read absorbance on a spectrophotometer within 5 minutes at 450 nm with a reference
wavelength of 655 nm. Blank the plate reader according to the manufacturer’s instructions
using the blank wells.

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10Preparation of Nuclear Extract
For your convenience, Active Motif offers a Nuclear Extract Kit (Cat. Nos. 40010 & 40410). This
kit contains buffers optiimized for use in TransAM Kits, which serves to reduce inconsistencies in
the assay that may arise from using homemade or other buffers. If you prefer to make your own
buffers, please refer to the following protocol.
This procedure can be used for a confluent cell layer of 75 cm2 (100-mm dish). The yield isapproximately 0.5 mg of nuclear proteins for 107 cells.
1.

Wash cells with 10 ml of ice-cold PBS/PIB.

2.

Add 10 ml of ice-cold PBS/PIB and scrape the cells off the dish with a cell lifter. Transfer thecells into a pre-chilled 15 ml tube and spin at 300 x g for 5 minutes at 4°C.

3.

Resuspend the pellet in 1 ml of ice-cold HB buffer by gentle pipetting and transfer the cells
into a pre-chilled 1.5 ml tube.

4.

Allow the cells to swell on ice for 15 minutes.

5.

Add 50 µl 10% Nonidet P-40 (0.5 % final) and mix by gentle pipetting.

6.

Centrifuge the homogenate for 30 seconds at 4°C in a microcentrifuge.

7.

Resuspend the nuclear pellet in 50 µl Complete Lysis Buffer and rock the tube gently on
ice for 30 minutes on a shaking platform.

8.

Centrifuge for 10 minutes at 14,000 x g at 4°C and save the supernatant (nuclear cell
extract). Aliquot and store at –80°C. Avoid freeze/thaw cycles.

9.

Determine the protein concentration of the extract by using a protein assay, such as Active
Motif’s ProStain™ Protein Quantification Kit (Cat. No. 15001). .

10X PBS
0.1 M phosphate buffer, pH 7.5
1.5 M NaCl
27 mM KCl

For 250 ml, mix:
3.55 g Na2HPO4 + 0.61 g KH2PO4
21.9 g
0.5 g

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.
PIB (Phosphatase Inhibitor Buffer)
125 mM NaF
250 mM β-glycerophosphate
250 mM para-nitrophenyl phosphate (PNPP)
25 mM NaVO3

For 10 ml, mix
52 mg
0.55 g1.15 g
31 mg

Adjust to 10 ml with distilled water. Mix the chemicals by vortexing. Incubate the solution at 50ºC
for 5 minutes. Mix again. Store at –20°C.

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11

PBS/PIB
Prior to use, add 0.5 ml of PIB to 10 ml of 1X PBS.
HB (Hypotonic Buffer)
20 mM Hepes, pH 7.5
5 mM NaF
10 µM Na2MoO4
0.1 mM EDTA

For 50 ml, mix
0.24 g
12 mg
5 µl of a 0.1 M solution
10 µl of a 0.5 M solution

Adjust pH to 7.5 with 1 N NaOH. Adjust volume to 50 ml with distilled water. Sterilize by filtering
through a 0.2 µm filter. Store the filter-sterilized solution at 4°C.

References
1.
2.
3.
4.

Rao A., Luo C. and Hogan P.G. (1997) Annu. Rev. Immunol. 15: 707-47.
Horsley V. and Pavlath G. K. (2002) J. Cell Biol. 156: 771-4.
Kiani A., Rao A. and Arambvuru J. (2000) Immunity 12: 359-72.
Jain J., Burgeon E., Badalian T.M., Hogan P.G. and Rao A. (1995) J. Biol Chem. 270: 413845.
5. Luo C., Burgeon E., Carew J.A., McCaffrey P.G., Badalian T.M., Lane W.S., Hogan P.G. and
Rao A. (1996) Mol. Cell Biol. 16: 3955-66.
6. Hoey T., Sun Y.L., Williamson K. and Xu X. (1995) Immunity 2: 461-72.
7. Chen L., Oakley M.G., Glover J.N., Jain J., Dervan P.B., Hogan P.G., Rao A. and Verdine G.L.
(1995) Curr. Biol. 5: 882-9.
8. Musaro A., McCullagh K.J., Naya F.J., Olson E.N. and Rosenthal N. (1999) Nature 400: 5815.
9. McCaffrey P.G., Goldfeld A.E. and Rao A. (1994) J. Biol. Chem. 269: 30445-50.
10. Alroy I., Towers T.L. and Freedman L.P. (1995) Mol. Cell Biol. 15: 5789-99.
11. Thompson C.B., Wang C.Y., Ho I.C., Bohjanen P.R., Petryniak B., June C.H., Miesfeldt S.,
Zhang L., Nabel G.J., Karpinski B., et al. (1992) Mol. Cell Biol. 12: 1043-53.
12. Wang C.Y., Bassuk A.G., Boise L.H., Thompson C.B., Bravo R., Leiden J.M. (1994) Mol. Cell
Biol. 14: 1153-9.
13. Renard P., Ernest I., Houbion A., Art M., Le Calvez H., Raes M. and Remacle J. (2001)
Nucleic Acids Res. 29: (4) e21.

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12

Appendix
Section A. Troubleshooting Guide
PROBLEMPOSSIBLE CAUSE

RECOMMENDATION

No signal or weak signal
in all wells

Omission of key reagent

Check that all reagents have been addedin the correct order

Substrate or conjugate is nolonger active

Test conjugate and substrate for activity

Enzyme inhibitor present

Sodium azide will inhibit the peroxidase
reaction, follow our recommendations to
prepare buffers

Plate reader settings not optimal

Verify the wavelength and filter settings in
the plate reader

Incorrect assay temperature

Bring substrate to room temperature

Inadequate volume ofDeveloping Solution

Check to make sure that correct volume
is delivered by pipette

Developing time too long

Stop enzymatic reaction as soon as the
positive wells turn medium-dark blue

Concentration of antibodies
too high

Increase antibody dilutions

Inadequate washing

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

High background in all wells

Uneven color development

High background in sample wells

No signal or weak signal insample wells

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Incomplete washing of wells
Ensure all wells are filled with Wash Buffer
and follow washing recommendations
Well cross-contamination

Follow washing recommendations

Too much nuclear extract per well

Decrease amount of nuclear extract down to
1-2 µg/well

Concentration of antibodies
too high

Perform antibody titration to determine
optimal working concentration. Start using
1:1000 for primary antibody and 1:5000 for thesecondary antibody. The sensitivity of theassay will be decreased

Not enough nuclear extract per well

Increase amount of nuclear extract not toexceed 40 µg/well

NFAT is poorly activated orinactivated in nuclear fractions

Perform a time course for NFAT activationin the studied cell line

Extracts are not from humanor mouse origin

Perform study with a human or mouse model

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:Direct:Fax:
E-mail:Active Motif Europe
UK Free Phone:France Free Phone:
Germany Free Phone:Direct:Fax:
E-mail:877.222.9543
760.431.1263
760.431.1351
tech_service@activemotif.com
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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