TransAM™ HIF-1(version F2)
Catalog Nos. 47096 & 47596

Copyright 2011 Active Motif, Inc.

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.
© 2011 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.

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TABLE OF CONTENTS

Page

Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1
Flow Chart of Process . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Introduction
HIF-1 Transcription Factor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Transcription Factor Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
TransAM HIF-1. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
Kit Performance and Benefits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Kit Components and Storage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
Additional Materials Required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
Protocols
Buffer Preparation and Recommendations. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
Quick Chart for Preparing Buffers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
HIF-1 Transcription Factor Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Preparation of Nuclear Extract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Appendix
Section A. Preparation of Nuclear Extract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Section B. Troubleshooting Guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Technical Services . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16

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Overview
Hypoxia-Inducible Factor 1 (HIF-1) is a transcription factor that is activated in response to low oxygen levels, which occur during hypoxic pulmonary hypertension, or cerebral and myocardial ischemia. Angiogenesis is the most common physiological response observed upon up-regulation of
HIF-1α. Therefore, accurate monitoring of HIF activation in cells, tissues or animals is crucial for
drug development and biomedical research. 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
with a sensitive and specific assay. TransAM HIF-1 Kits are designed specifically for the study of
HIF-1. They contain a 96-well plate to which oligonucleotide containing the hypoxia response
element (HRE) has been immobilized. HIF dimers contained in nuclear extracts bind specificallyto this oligonucleotide and are detected through the use of an antibody directed against HIF-1α.
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 HIF-1 Kits are available in two sizes:

product

formatcatalog no.

TransAM™ HIF-1

1 x 96 rxns

47096

TransAM™ HIF-1

5 x 96 rxns

47596

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

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1Flow Chart of Process

2

1

5

4

3

7

6

8

9

10

11

12

A
B
C
D
E

Cell extractcontaining activatedtranscription factor

F
G
H

Oligonucleotide
coated plate

Add cell
extract
21

11

01

9

8

7

6

5

4

3

2

1
A
B

C
D
E
F
G
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Add primary
antibody

1 hr.
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2

1

9

8

7

6

5

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10

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12

A
B
C
D
E
F
G
H

Add anti-IgG
HRP conjugate
21

11

01

9

8

7

6

5

4

1 hr.
3

2

1
A
B

C
D
E
F
G

Add developing
and stop solution

H

1 hr.
1

5

4

3

2

7

6

A
B
C
D
E
F
G
H

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Introduction
HIF-1 Transcription Factor
All organisms have mechanisms for sensing oxygen concentrations and for responding to low
oxygen levels (hypoxia) with changes in gene expression. The transcription factor HypoxiaInducible Factor 1 (HIF-1) is one of the key regulators of oxygen homeostasis1. It is required for thedevelopment of key physiological systems, such as vasculogenesis and pneumogenesis, during
fetal and postnatal life. HIF-1 also regulates the physiological responses to hypoxia and thepathophysiology of heart attack, cancer, stroke and chronic lung disease (see 2 for review).
HIF-1 is a heterodimeric protein that consists of two subunits, HIF-1α and HIF-1β (also known as
ARNT1, the aryl hydrocarbon nuclear translocator)3. They belong to a larger family of transcription
factors that contain basic helix-loop-helix (bHLH) and PER-ARNT-SIM homology domains2. ClassI members of this family such as HIF-1α, HIF-2α and HIF-3α heterodimerize with one of the class
II sub-family such as ARNT1, ARNT2 and ARNT3, resulting in different DNA-binding and transcriptional properties depending on the dimer formation. HIF-1α and HIF-1β mRNA are expressed inmost human and rodent tissues4. HIF-2α, HIF-3α, ARNT2 and ARNT3 are restricted to certain
tissues, and therefore have a more specialized role in oxygen homeostasis.
Whereas HIF-1β is constitutively expressed, the expression of HIF-1α is induced by hypoxia(oxygen concentration below 6%)5. Under nonhypoxic conditions, HIF-1α is hydroxylated by a
prolyl hydroxylase enzyme6. This modification is required for the binding of the von Hippel-Lindau(VHL) protein to HIF-1α and its subsequent ubiquitination and degradation in the proteasome7.
Iron chelators and cobalt chloride prevent HIF-1α ubiquitination besides inducing its expression8.
Therefore these chemicals have been used in mammalian cells to generate HIF study models. HIF
heterodimers bind to the hypoxia response element (HRE), a 5´-RCGTG-3´ consensus sequence9.
Several dozen HIF-1-regulated genes have been identified so far, including genes coding for proteins involved in angiogenesis, energy metabolism, erythropoiesis, cell proliferation and viability,
vascular remodeling and vasomotor responses (see 2 for review).
Transcription Factor Assays
To date, three methods are widely used to measure HIF-1 activation, either directly or indirectly:
1. Western blot: HIF-1 activation can be determined by Western Blot by using antibodies
specific for HIF-1α 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. Gel retardation: The DNA-binding capacity of HIF-1 can be assayed by gel retardation, also
called electrophoretic mobility shift assay (EMSA). In this case, nuclear extracts are incubated with a radioactive double-stranded oligonucleotide probe containing the consensus
sequence for HIF binding. If HIF-1 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. Gel-shift assays also require special
precautions and equipment for handling radioactivity.
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33. Reporter assays: Another largely used method to assay HIF-1 activation is based on reporter genes, typically luciferase or β-galactosidase, placed under the control of a promoter
containing a HRE. The promoter can be artificial, made of several HRE cis-elements and a
TATA box, or natural, like the erythropoietin promoter sequence. However, the procedure islimited by the following issues: (i) reporter gene assays have to be repeated several times
to obtain 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 perform with a large number of samples, but require efficient cell transfection with thereporter plasmid..

TransAM HIF-1
HIF-1 is involved in every major cause of mortality in the industrialized world such as heart attack,cancer, stroke and chronic lung disease, and therefore represents an excellent pharmacologicaltarget. 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 HIF-1 activation10. TransAM Kits combine a fast, user-friendly ELISA format with a sensitive and specific assay
for transcription factors. TransAM HIF-1 Kits contain a 96-well plate on which has been immobilized an oligonucleotide containing a HRE (5´-TACGTGCT-3´) from the EPO gene. HIF contained innuclear extract specifically binds to this oligonucleotide. The primary antibody used in TransAM
HIF-1 Kits recognizes an epitope on HIF-1α protein that is accessible 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 the assay is performed in 96-well plates, a large number of samples
can be handled simultaneously, enabling high-throughput automation. The assay is specific for
HIF-1α activation and has been shown to be more sensitive and faster than the gel-retardation
technique. With the 3.5-hour TransAM procedure, we could detect HIF-1 activation with as little as
2.5 µg of nuclear extract from CoCl2-treated cells. A comparative assay using EMSA required 5µg of nuclear extract and a 3-day autoradiography.
TransAM has many applications including the study of drug potency toward HIF-1-related
enzymes, HIF-1 transcriptional activity regulation and protein structure/function of HIF-1α and its
cofactors in areas such as angiogenesis, erythropoiesis, tumorigenesis and embryogenesis.

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4Kit Performance and Benefits
The TransAM HIF-1 Kit is for research use only. Not for use in diagnostic procedures.
Detection limit: > 2.5 µg nuclear extract/well.
Range of detection: TransAM provides quantitative results within a 2.5 - 20 µg nuclear extract/well range (see graph below).
Cross-reactivity: TransAM HIF-1 detects HIF-1α in human and monkey extracts. It does not crossreact with other species or HIF subunits.
Assay time: 3.5 hours. TransAM is 20-fold faster than EMSA.

HIF-1 alpha activation (OD 450 nm)

0.35
0.3

HeLa

0.25

HeLa + CoCl2

0.2
0.15
0.1
0.05
0
1.25

2.5

5

10

20

Nuclear extract (µg/well)
Monitoring HIF-1α activation with TransAM HIF-1 Kit.
Different amounts of nuclear extract from untreated and CoCl2-treated HeLa cells are tested for HIF activation using theTransAM HIF-1 Kit. This data is provided for demonstration only.

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5Kit Components and Storage
TransAM HIF-1 Kits are for research use only. Not for use in diagnostic procedures. The nuclear
extract must be kept at -80°C. Other Kit components can be stored at -20°C prior to first use.
Then, we recommend storing each component at the temperature indicated in the table below.
All components are guaranteed stable for 6 months from date of receipt when stored properly.
Quantity
1 plate / 5 plates

Storage

HIF-1 antibody (0.25 mg/ml)

22 µl / 110 µl-20°C for 6 months

Anti-mouse HRP-conj. IgG (0.4 mg/ml)

11 µl / 55 µl

4°C for 6 months

Wild-type oligonucleotide AM13
(10 pmol/µl)

100 µl / 500 µl-20°C for 6 months

Mutated oligonucleotide AM13
(10 pmol/µl)

100 µl / 500 µl-20°C for 6 months

HeLa (CoCl2) nuclear extract (2.5 mg/ml)

50 µl / 250 µl-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

Lysis Buffer AM1

10 ml / 50 ml

4°C

Binding Buffer AM4

10 ml / 50 ml

4°C

10X Wash Buffer AM2

22 ml / 110 ml

4°C

10X Antibody Binding Buffer AM3

2.2 ml / 11 ml

4°C

Developing Solution

11 ml / 55 ml

4°C

Stop Solution

11 ml / 55 ml

4°C

Reagents

96-well IRF-3 assay plate

1/5

Plate sealer

1/5

Additional materials required• Multi-channel pipettor• Multi-channel pipettor reservoirs• Rocking platform• Microplate spectrophotometer capable of reading at 450 nm (655 nm as optional reference
wavelength)www.activemotif.com

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). The presence of phosphatase inhibitors gives a yellow coloration to Lysis Buffer AM1.
Prepare the amount of Complete Lysis Buffer required for the assay (see the Quick Chart for
Preparing Buffers in this section) by adding 1 µl 1 M DTT and 10 µl Protease Inhibitor Cocktail per
ml of Lysis Buffer AM1. 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 (see the Quick Chart for
Preparing Buffers in this section) by adding 1 µl 1 M DTT per ml of Binding Buffer AM4. After use,
discard remaining Complete Binding Buffer.
Preparation of 1X Wash Buffer
Prepare the amount of 1X Wash Buffer required for the assay (see the Quick Chart for Preparing
Buffers in this section) as follows: For every 100 ml of 1X Wash Buffer required, dilute 10 ml 10X
Wash Buffer AM2 with 90 ml of 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 the 10X Wash Buffer AM2 mayform clumps, therefore homogenize the buffer by incubating at 50°C 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 (see the Quick Chartfor Preparing Buffers in this section*) as follows: For every 10 ml of 1X Antibody Binding Buffer
required, dilute 1 ml 10X Antibody Binding Buffer AM3 with 9 ml of distilled water. 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
to room temperature and vortexing for 1 minute prior to use. Dilute the primary antibody to 1:500
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 the 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 (see the Quick Chart for
Preparing Buffers in this section) into a secondary container. After use, discard remaining Stop
Solution.
WARNING: The Stop Solution is corrosive. Wear personal protective equipment whenhandling, i.e. safety glasses, gloves and labcoat.
HeLa nuclear extract
The HeLa (CoCl2) nuclear extract is provided as a positive control for HIF-1 activation. Sufficient
extract is supplied for 10 reactions per plate. This extract is optimized to give a strong signal
when used at 10 µg/well. We recommend aliquoting the extract in 10 µl fractions and storing at-80°C. 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 HIF binding in order tomonitor the specificity of the assay. Used at 20 pmol/well, the oligonucleotide will prevent HIF
binding to the probe immobilized on the plate. Conversely, the mutated consensus oligonucleotide should have no effect on HIF binding. Prepare the required amount of wild-type and/or
mutated consensus oligonucleotide by adding 2 µl of the appropriate oligonucleotide to 43 µl
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

1 well

1 strip(8 wells)

6 strips(48 wells)

12 strips(96 wells)

Complete Lysis Buffer

DTT
Protease Inhibitor Cocktail
Lysis Buffer
Total Required

0.01 µl0.12 µl11.12 µl11.25 µl

0.1 µl0.9 µl89.0 µl90.0 µl

0.6 µl5.4 µl534.0 µl
540.0 µl

1.2 µl10.8 µl1.07 ml
1.08 ml

Complete Binding Buffer

DTT
Binding Buffer
Total Required

0.04 µl44.5 µl
45 µl

0.3 µl356.1 µl
360 µl2.16 µl2.14 ml
2.16 ml

4.3 µl4.27 ml
4.32 ml

Binding Buffer with
IRF wt or mut oligont

wt or mut oligont
Complete Binding Buffer
Total Required

2.0 µl43.0 µl45.0 µl

16.0 µl344.0 µl360.0 µl96.0 µl
2.064 µl2.16 µl

N/A
N/A
N/A

1X Washing Buffer

Distilled Water
10X Washing Buffer
Total Required

2.025 ml
225.0 µl2.25 ml

16.2 ml
1.8 ml18.0 ml97.2 ml
10.8 ml
108.0 ml

194.4 ml21.6 ml
216.0 ml

1X Antibody
Binding Buffer*

Distilled Water
202.5 µl
10X Antibody Binding Buffer 22.5 µl
Total Required
225.0 µl

1.62 ml
180.0 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 µl900.0 µl

5.4 ml10.8 ml

Stop Solution

Total Required

112.5 µl900.0 µl

5.4 ml10.8 ml* Volumes listed refer to the preparation of buffer for diluting both the primary & secondary
antibodies.

HIF-1 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 HIF-1 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 that contains 20 pmol (2 µl)of the wild-type or mutated 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 2-20 µg of nuclear extract diluted in Complete Lysis Buffer per well. A protocol
for preparing nuclear extracts is provided on page 11.
Positive control wells: Add 10 µg of the provided nuclear extract diluted in 10 µl of Complete Lysis Buffer per well (4 µl of nuclear extract in 6 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 HIF-1 antibody (1:500 dilution in 1X Antibody Binding Buffer) to each wellbeing 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).

1.

Add 100 µl of diluted 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).

1.

Add 100 µl Developing Solution to all wells being used.

2.

Incubate 8-12 minutes at room temperature protected from direct light. 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.

Step 3: Binding of secondary antibody

Step 4: Colorimetric reactionwww.activemotif.com

10References
1.

Richard D.E., Berra E. and Pouysségur J. (1999) Biochem. Biophys. Res. Commun. 266: 71822.
2. Semenza G.L. (2000) J. Appl. Physiol. 88: 1474-80.
3. Wang G.L., Jiang B.H., Rue E.A. and Semenza G.L. (1995) Proc. Natl. Acad. Sci. USA 92:
5510-4.
4. Wiener C.M., Booth G. and Semenza G.L. (1996) Biochem. Biophys. Res. Commun. 225:
485-8.
5. Jiang B.H., Semenza G.L., Bauer C. and Marti H.H. (1996) Am. J. Physiol. 271: C1172-80.
6. Zhu H. and Bunn H.F. (2001) Science 292: 449-51.
7. Tanimoto K., Makino Y., Pereira T. and Poellinger L. (2000) EMBO J. 19: 4298-4309.
8. Wang G.L. and Semenza G.L. (1993) Blood 82: 3610-5.
9. Semenza G.L., Jiang B.H., Leung S.W., Passantino R., Concordet J-P., Maire P. and Giallongo
A. (1996) J. Biol. Chem. 271: 32529-37.
10. Renard P., Ernest I., Houbion A., Art M., Le Calvez H., Raes M. and Remacle J. (2001) Nucleic
Acids Res. 29: (4) e21.

Appendix
Section A. 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 confluent cell layer of 75 cm2 (100-mm dish). The yield is approximately 0.15 mg of nuclear proteins for 9 x 106 cells.
1.

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

2.

Add 10 ml ice-cold PBS/PIB and scrape the cells off the dish with a cell lifter. Transfer cells
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 ice-cold HB buffer by gentle pipetting and transfer the cells intoa 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 vortex the tube vigorously for 10 seconds.

6.

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.)

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 extract).
Aliquot and store at –80°C. Avoid freeze/thaw cycles.
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11

9.

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

Preparation of Buffers for Nuclear Extract
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

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

For 10 ml, mix:
52 mg

250 mM β-glycerophosphate

0.55 g

250 mM p-nitrophenyl phosphate (PNPP) 1.15 g
25 mM NaVO3

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 PIB to 10 ml 1X PBS.
HB (Hypotonic Buffer)

For 50 ml, mix:

20 mM Hepes, pH 7.5

0.24 g

5 mM NaF

12 mg

10 µM Na2MoO4

5 µl of a 0.1 M solution

0.1 mM EDTA

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.

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12Section B: Troubleshooting Guide
Problem/question

Possible cause

Recommendation

No signal or weak signal

Omission of key
reagent

Check that all reagents have been added in all wells in thecorrect order

Substrate or conjugate
is no longer active

Test conjugate and substrate for activity

Enzyme inhibitor
present

Sodium azide will inhibit the peroxidase reaction, follow ourrecommendations 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 is too highIncrease antibody dilutions

Inadequate washing

Ensure all wells are filled with Wash Buffer and follow washingrecommendations

Incomplete washingof wells

Ensure all wells are filled with Wash Buffer and follow washingrecommendations

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 is too high

Perform antibody titration to determine optimal working concentration. Start using 1:1000 for primary antibody and 1:5000
for the secondary antibody. The sensitivity of the assay will bedecreased

Not enough nuclear
extract per well

Increase amount of nuclear extract to 50 µg/well

HIF is poorly activated or inactivated innuclear fractions

Perform a time course for HIF activation in the studied cell line

High background in all
wells

Uneven color development

High background in sample
wells

No signal or weak signal insample wells

Nuclear extracts
are not from correct
specieswww.activemotif.com

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

www.activemotif.com

14