TransAM™

PPARγ Transcription Factor
Assay Kits
(version E2)
Catalog Nos. 40196 & 40696

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 this manual at any
time.
This documentation may not be copied, transferred, reproduced, disclosed, or duplicated, inwhole 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 and international treaties.
The manufacturer of this documentation is Active Motif, Inc.
© 2007 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 their respective
companies.

TABLE OF CONTENTS

Page

Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Flow Chart of Process . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Introduction
PPARγ Transcription Factor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Transcription Factor Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
TransAM PPARγ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
Kit Performance and Benefits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Kit Components and Storage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
Protocols
Buffer Preparation and Recommendations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
Quick Chart for Preparing Buffers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
PPARγ 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
Peroxisome proliferator-activated receptor γ (PPARγ) is a transcription factor that is activated in
response to a high-fat diet. Besides its key role in lipid metabolism, PPARγ has been shown to
be involved in chronic diseases such as diabetes, obesity, atherosclerosis and cancer. Therefore,
accurate monitoring of PPARγ activation in cells, tissues or animals is crucial for biomedical research and drug development. To date, such research 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 witha sensitive, specific assay. TransAM PPARγ Kits are designed specifically for the study of PPARγ.
They contain a 96-well plate to which oligonucleotide containing the peroxisome proliferator
response element (PPRE) has been immobilized. PPARs contained in nuclear extracts bind specifically to this oligonucleotide and are detected through use of an antibody directed against PPARγ.
Addition of a secondary antibody conjugated to horseradish peroxidase (HRP) provides sensitive
colorimetric readout that is easily quantified by spectrophotometry. The 96-well plate with individual strips of 8 wells is suitable for manual use or for high-throughput screening applications.
TransAM PPARγ Kits are available in two sizes:
product
TransAM PPARγ

formatcatalog no.

1 x 96-well plate5 x 96 well plates

40196
40696

See Active Motif products related to the PPARγ 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
PPARγ Transcription Factor
Peroxisome proliferator-activated receptors (PPARs) are nuclear receptors involved in lipid
transport and metabolism (see 1 for review). They form a subfamily of the nuclear receptor
superfamily, along with the receptors for thyroid hormone, retinoic acid, vitamin D, ecdysone andthe orphan receptors Rev-ErbAα and E75. Three PPAR isotypes have been identified: α, β (alsocalled δ and NUC1) and γ. Their roles in chronic diseases such as diabetes, obesity, atherosclerosis and cancer are heavily investigated2, 3. PPARα and PPARγ contribute to atherosclerotic
plaque formation by regulating plasma lipoprotein concentrations, affecting foam cell formation,
modulating the inflammatory response and influencing plaque stability. PPARα acts as a global
regulator of fatty acid catabolism. Recent studies have shown that PPARβ is also involved inlipid metabolism. PPARγ can modulate inflammation by reducing the production of inflammatory
cytokines in monocytes4, 5. PPARγ may also promote atherosclerosis by stimulating the uptake ofoxidized LDL that supports foam cell formation6. In contrast to PPARα, PPARγ promotes fat storage by increasing adipocyte differentiation and transcription of a number of lipogenic proteins.
PPARα is expressed mostly in brown adipose tissue and liver, then kidney, heart and skeletal
muscle. PPARγ is mainly expressed in adipose tissue and, to a lesser extent, in the colon, theimmune system and the retina. PPARβ is found in many tissues, but the highest expression is in
the gut, kidney and heart.
PPARs are activated by natural ligands such as fatty acids and eicosanoids, and various synthetic
ligands such as lipid-lowering fibrates and anti-diabetic glitazones7, 8. PPARs bind to peroxisome
proliferator response elements (PPREs), which consist of a direct repeat of the nuclear receptor
hexameric DNA core recognition motif spaced by one nucleotide (5´-AACTAGGNCAAAGGTCA-3´).
PPARs bind only to PPREs as heterodimers with the receptor for 9-cis retinoic acid RXR (retinoid
X receptor). The PPRE repeat structure imposes a polarity to the bound heterodimer9. PPAR interacts with the upstream extended core hexamer, whereas RXR occupies the downstream motif.
PPAR-mediated transactivation results from the combination of PPAR:RXR binding to a PPRE andligand activation of this complex. The conformational change of PPAR triggered by ligand binding,or by other activation processes such as phosphorylation, is believed to generate a transcriptionally active complex by forming specific contacts with coactivator proteins.

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

PPARγ activation can be determined by Western Blot by using antibodies specific for PPARγ
protein. 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 PPARγ 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 sequencewww.activemotif.com

3

for PPAR binding. If PPARγ is active in the nuclear extract, it 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)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 PPARγ activation is based on reporter genes, typicallyluciferase or β-galactosidase, placed under the control of a promoter containing a PPRE.
The promoter can be artificial, made of several PPRE cis-elements and a TATA box, or natural, like the acylCoA oxidase promoter sequence. However, the procedure is limited by
the following issues: (i) reporter gene assays have to be repeated several times to obtainstatistically reliable data; and (ii) reporter gene assays are sensitive to confounding factorsthat 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 perform with alarge number of samples, but require efficient cell transfection with the reporter plasmid.

TransAM PPARγ
PPARγ is involved in fatty acid metabolism and intake by adipocytes, and therefore represents an
excellent pharmacological target to develop drugs for treating obesity and diabetes. However,pharmaceutical research in this field has been hampered by the lack of convenient assays suitable for high sample number experiments.
To overcome this, Active Motif is introducing a high-throughput assay to quantify PPARγ activation10. The TransAM Kit combines a fast and user-friendly ELISA format with a sensitive andspecific assay for transcription factors. TransAM PPARγ Kits contain a 96-well plate on which has
been immobilized an oligonucleotide that contains a PPRE (5´-AACTAGGTCAAAGGTCA-3´). PPAR
contained in nuclear extract specifically binds to this oligonucleotide. The primary antibody usedin the TransAM PPARγ Kit recognizes an accessible epitope on PPARγ protein upon DNA binding.
Addition of a secondary HRP-conjugated antibody provides a sensitive colorimetric readout easily
quantified by spectrophotometry. Once the nuclear extracts are prepared, this assay is completed
in less than 3.5 hours. As this assay is performed in 96-well plates, a large number of samples
can be handled simultaneously, enabling high-throughput automation. This assay is specific for
PPARγ activation and has been shown to be 5-fold more sensitive and 20-fold faster than the
gel-retardation technique. With the 3.5-hour TransAM procedure, we could detect PPARγ activation using as little as 1.0 µg of nuclear extract. A comparable assay using EMSA required 5 µg of
nuclear extract and a 3-day autoradiography.
TransAM has many applications including the study of drug potency toward PPARγ binding,
PPARγ transcriptional activity regulation and protein structure/function studies of PPARγ and its
cofactors in areas such as diabetes, obesity, atherosclerosis, carcinogenesis, and many more.

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4Kit Performance and Benefits
The TransAM PPARγ Kit is for research use only. Not for use in diagnostic procedures.
Detection limit: > 1.0 µg nuclear extract/well. TransAM PPARγ is 5-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 PPARγ detects PPARγ1 and PPARγ2 from human, mouse and rat origins.
It will not cross-react with PPARα or PPARd.
Assay time: 3.5 hours. TransAM is 20-fold faster than EMSA.

1.2
1

COS-7
COS-7 (PPARγ transfected)

0.8
0.6
0.4
0.2
0
0.63

1.25

2.5

5

10

-0.2

Nuclear Extract (µg/well)
Monitoring PPAR activation with the TransAM PPARγ Kit: Different amounts of nuclear extracts fromuntransfected and PPARγ transfected COS-7 cells are tested for PPAR activation by using the TransAM PPARγ
Kit. These results are 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.
Quantity
1 plate / 5 plates

Storage / Stability

PPARγ antibody

12 µl /60 µl-20°C for 6 months

Anti-mouse HRP-conjugated IgG

11 µl / 55 µl(0.2 mg/ml)-20°C for 6 months

Wild-type oligonucleotide AM8

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

Mutated oligonucleotide AM8

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

COS-7 nuclear extract (PPARγ transfected)

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

Dithiothreitol (DTT)(1 M)

100 µl / 500 µl-20°C for 6 months

Protease Inhibitor Cocktail

100 µl / 500 µl-20°C for 6 months

Herring sperm DNA

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

Lysis Buffer AM1

10 ml / 50 ml

4°C for 6 months

Binding Buffer AM6

10 ml / 50 ml

4°C for 6 months

10X Wash Buffer AM2

22 ml / 110 ml

4°C for 6 monthsl

10X Antibody Binding Buffer AM2

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 PPARγ assay plate

1/5

4°C for 6 months

Plate sealer

1/5

Reagents

Additional materials required

For Nuclear Extract preparation• Multi-channel pipettor• Hypotonic Buffer• Multi-channel pipettor reservoirs• Phosphatase Inhibitor Buffer• Rocking platform• 10X PBS

• Microplate spectrophotometer capable of 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 nuclear 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 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 Buffer immediately for cell
lysis. The remaining amount should be discarded if not used in the same day.
Preparation of Complete Binding Buffer
Prepare the amount of Complete Binding Buffer required for the assay by adding 10 µl of 1 mg/
ml Herring sperm DNA per ml of Binding Buffer AM6 (see the Quick Chart for Preparing Buffers inthis section). After use, discard remaining Complete Binding Buffer.
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 formclumps, therefore homogenize the buffer by incubating at 50ºC for 2 minutes and mixing prior touse.
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 toroom temperature and vortexing for 1 minute prior to use. Dilute the primary antibody to 1:1000
and the HRP-conjugated secondary antibody to 1:1000 with the 1X Antibody Binding Buffer. Depending on the particular assay, the signal:noise ratio may be optimized by using higher dilutionsof 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 at room
temperature for at least 1 hour. Transfer the amount of Developing Solution required for the 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 COS-7 nuclear extract (PPARγ transfected) is provided as a positive control for PPARγ
activation. Sufficient extract is supplied for 8 reactions per plate. This extract is optimized to givea strong signal when used at 5 µg/well. We recommend aliquoting the extract in 5 µl fractions
and storing at -80ºC. Avoid multiple freeze/thaw cycles of the extract. Various cell extracts areavailable 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 PPARγ binding in orderto monitor the specificity of the assay. Used at 40 pmol/well, the oligonucleotide will prevent
PPARγ binding to the probe immobilized on the plate. Conversely, the mutated consensus oligonucleotide should have little effect on PPARγ binding. Prepare the required amount of wild-type
and/or mutated consensus oligonucleotide by adding 2 µl of appropriate oligonucleotide to 43 µlof Complete Binding Buffer 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 nuclear extract.

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8Quick Chart for Preparing Buffers
Reagents to prepare

Components

For 1
well

For 1 strip(8 wells)

For 6 strips(48 wells)

For 12 strips(96 wells)

Complete Lysis Buffer

DTT
Protease Inhibitor Cocktail
Lysis Buffer AM1
TOTAL REQUIRED

0.01 µl0.12 µl11.12 µl11.25 µl

0.1 µl0.9 µl
89 µl
90 µl

0.6 µl5.4 µl534 µl540 µl1.2 µl10.8 µl
1.068 ml
1.08 ml

Complete Binding Buffer

Herring sperm DNA
Binding Buffer AM6
TOTAL REQUIRED

0.45 µl44.5 µl
45 µl

3.6 µl356 µl360 µl21.6 µl2.138 ml
2.16 ml43.2 µl
4.277 ml
4.32 ml

Complete Binding Buffer
with wild-type ormutated oligonucleotide

Wild-type or mutated oligo
Complete Binding Buffer
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
216 ml1X Antibody
Binding Buffer*

Distilled water
10X Ab Binding Buffer AM2
TOTAL REQUIRED

202.5 µl22.5 µl
225 µl1.62 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* Volumes listed refer to the preparation of buffer for diluting both the primary & secondary antibodies.

PPARγ 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, Complete Binding Buffer, 1X Wash Buffer and 1X Antibody
Binding Buffer as described above in the section Buffer Preparation and Recommendations.
Multi-channel pipettor reservoirs may be used for dispensing the Complete 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 PPARγ to its consensus sequence1.

Add 40 µl Complete Binding Buffer to each well to be used. If you wish to perform competitive binding experiments, add 40 µl Complete Binding Buffer containing 40 pmol (2 µl)of the wild-type or mutated consensus oligonucleotide (see the Buffer Preparation section
above for a description of 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 PPARγ antibody (1:1000 dilution in 1X Antibody Binding Buffer) to allwells 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 reaction1.

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 optimized 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 15 ml cell suspension in a T75 flask. The yield is approximately
50 µg of nuclear proteins for 107 cells.
1.

Collect 10 ml of cell suspension in a pre-chilled 15 ml tube.

2.

Scrape the cells off the flask in the remaining 5 ml of media with a cell lifter. Transfer cells
into the 15 ml tube and spin at 300 x g for 5 minutes at 4°C.

3.

Discard supernatant. Resuspend cell pellet in 5 ml PBS/PIB and spin at 300 x g for 5 minutes at 4°C.

4.

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

5.

Allow the cells to swell on ice for 15 minutes.

6.

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

7.

Centrifuge the homogenate for 30 seconds at 4°C in a microcentrifuge. Remove the supernatant (cytoplasmic fraction) and, if you wish to save this for other uses, transfer it into a
pre-chilled microcentrifuge tube. (Store the cytoplasmic fraction at –80°C.)

8.

Resuspend the nuclear pellet in 40 µl Complete Lysis Buffer and rock the tube gently on ice
for 30 minutes on a shaking platform.

9.

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

10. Determine the protein concentration of the extract by using a Bradford-based assay.

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.

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11

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.
PBS/PIB
Prior to use, add 0.5 ml of PIB in 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.
5.
6.
7.

Desvergnes B. and Wahli W. (1999) Endocrine Rev. 20: 649-88.
Fajas L, Debril M-B. and Auwerx J. (2001) J. Mol Endocrin. 27: 1-9
Kersten S., Desvergnes B. and Wahli W. (2000) Nature 405: 421-4.
Ricote M., Li A.C., Wilson T.M., Kelly C.J. and Glass C.K. (1998) Nature 391: 79-82.
Jiang C., Ting A.T. and Seed B. (1998) Nature 391: 82-86.
Nagy L., Tontonoz P., Alvarez J.G.A., Chen H. and Evans R.M. (1998) Cell 93: 229-240.
Lehmann J.M., Moore L.B., Smith-Oliver T.A., Wilkison W.O., Willson T.M. and Kliewer S.A.
(1995) J. Biol. Chem 270: 12953-6.
8. Forman B.M., Chen J. and Evans R.M. (1997) Proc. Natl. Acad. Sci. USA 94: 4312-7.
9. Ijpenberg A., Jeannin E., Wahli W. and Desvergne B. (1997) J. Biol. Chem. 272: 20108-17.
10. 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
1:500 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

PPARγ is poorly activated orinactivated in nuclear fractions

Perform a time course for PPARγ activationin the studied cell line

Nuclear extracts are not from rat,mouse or human origin

Perform study with a human, mouse or rat
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
Active Motif China
Direct: (86)-21-20926090
Cell Phone: 18521362870
E-mail: techchina@activemotif.com

Visit Active Motif online at activemotif.com