TransAM™

Sp1 & Sp1/Sp3
Transcription Factor Assay Kits
(version C4)
Catalog Nos. 41296 & 41796 (Sp1)
Catalog Nos. 40496 & 40996 (Sp1/Sp3)

TABLE OF CONTENTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Page
Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1
Flow Chart of Process . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Introduction
Sp1 and Sp3 Transcription Factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Transcription Factor Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
TransAM Sp1 & Sp1/Sp3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
Kit Performance and Benefits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Kit Components and Storage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
Protocols
Buffer Preparation and Recommendations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
Quick Chart for Preparing Buffers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Sp1 & Sp1/Sp3 Transcription Factor Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Preparation of Nuclear Extract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
Troubleshooting Guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Technical Services . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .14

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Overview
Sp1 is one of the first cellular transcription factors to be identified, cloned and characterized for
its ability to bind to GC-boxes in regulatory promoter elements. Sp1 has always been described
as a ubiquitous transcription factor that is required for the constitutive and inducible expressionof a variety of genes, such as in cell cycle or mammalian development. Identification of new
members of the Sp family, e.g. Sp2, Sp3, Sp4 and BTEB, have redefined the understanding of
Sp1-regulated genes. Therefore, accurate monitoring of Sp family activity 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 todetect and quantify transcription factor activation. TransAM Kits combine a fast, user-friendly
format with a sensitive, specific assay. TransAM Sp1 and Sp1/Sp3 Kits are designed specificallyfor the study of Sp1-regulated genes. They contain a 96-well plate to which an oligonucleotide
containing the Sp1 consensus binding site has been immobilized. Sp1 and Sp3 contained innuclear extracts bind specifically to this oligonucleotide and are detected through use ofan antibody directed against Sp1 or Sp3. Addition of a secondary antibody conjugated tohorseradish peroxidase (HRP) provides sensitive colorimetric readout that is easily quantified byspectrophotometry. The 96-well plate with individual strips of 8 wells is suitable for manual use
or for high-throughput screening applications. TransAM Sp1 and Sp1/Sp3 Kits are available in two
sizes:
product

formatcatalog no.

TransAM Sp1

1 x 96-well plate5 x 96 well plates

41296
41796

TransAM Sp1/Sp3

1 x 96-well plate5 x 96 well plates

40496
40996

The procedure outlined in this manual can be used for either TransAM Sp1 or TransAM Sp1/Sp3
Kits. See Active Motif products related to the Sp1 and Sp3 transcription factors 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
Sp1 and Sp3 Transcription Factors
The Sp1 transcription factor is a 105 kDa protein that can activate a wide subset of mammaliangenes containing upstream promoter elements called a GC box (GGGGCGGGG) and the related
GT/CACCC box (GGTGTGGGG)1, 2. The C-terminal domain of Sp1 harbors three contiguous
Cys-X4-Cys-X12-His-X3-His repeats, which are typical of the Cys2His2-type zinc-finger DNAbinding domain that was first found in the TFIIIA transcription factor. Specific variations in theubiquitous expression of Sp1 suggest its involvement in gene regulation of cell cycle, hormonal
activation and development patterning3. Sp1 knock-out embryos show a broad range ofabnormalities and usually die around day 11 of gestation. Recent studies suggest that Sp1 is an
important regulator of expression of the methyl-CpG-binding protein MeCP24.
The Sp zinc finger transcription factor family is composed of four members (Sp1, Sp2, Sp3 andSp4) that share similarity within their DNA-binding domains, transactivation domains and tissue
expression patterns2. Sp1, Sp3 and Sp4 are more closely related to each other than to Sp2,
which does not bind to a GC-box but to a GT-rich element5. Sp1 contains two glutamine-richtranscriptional activation domains that mediate direct interactions with the TATA box-binding protein (TBP) involved in the TFIID-RNA polymerase II complex. These activation domains can alsointeract directly with TAF110 transcription factor6. A cooperative interaction between Sp1 and
NFκB p65 is required for the efficient stimulation of HIV-1 transcription7. Sp1 has been shown tointeract with YY1, Oct-1, E2F-1, E2F-3 and p748, 9. The role of Sp3 alone is not well defined as
an activator or repressor. However, gene transcription has been shown to be regulated by theratio of Sp1 and Sp3 in different cell models10-12. These two Sp-family members compete witheach other to bind to Sp1 DNA binding sites. Sp4 expression appears to be restricted to a few
tissues. Sp4 is highly expressed during the development of the mouse embryo central nervous
system (CNS) and seems to be required for normal male reproductive behavior13. Recently, therole of Sp1 in breast cancer and aging has been reassessed due to its effect on estrogen andprogesterone receptor transcription levels14. Sp1 phosphorylation has also been the focus ofinvestigations on proapoptotic and angiogenic gene transcription regulation in vascular smoothmuscle and vascular endothelial cells15, 16.

Transcription Factor Assays
To date, three methods are widely used to measure Sp1/Sp3 expression, either directly or
indirectly:
1.

Sp1/Sp3 expression can be determined by Western blot by using antibodies specific for Sp1or Sp3 protein. This method is time consuming (up to 2 days once the nuclear cell extracts
are prepared), and is not suitable for processing large numbers of samples.

2.

The DNA-binding capacity of Sp1 and Sp3 can be assayed by gel retardation, also called
electrophoretic mobility shift assay (EMSA). In this method, nuclear extracts are incubatedwith a radioactive double-stranded oligonucleotide probe containing the consensus
sequence for Sp1 binding. If Sp1 and Sp3 are active in the nuclear extract, it will bind towww.activemotif.com

3the probe. Samples are then resolved by electrophoresis on a native polyacrylamide gel,followed by autoradiography. This method is sensitive, but like the previous procedure,
it is time consuming (multiple days of gel exposure may be required to achieve sufficientsensitivity) 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 Sp1 and Sp3 activation is based on reporter genes, typicallyluciferase or β-galactosidase, placed under the control of a promoter containing a Sp1
consensus binding site. The promoter can be artificial, made of a GC box and a TATA box,
or natural, like promoter sequences from viral regulator elements, such as the HIV-1 LTR
promoter. However, the procedure is limited by the following issues: (i) reporter geneassays 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 levelof 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 the reporter plasmid.

TransAM Sp1 & Sp1/Sp3
Sp1-regulated genes are involved in a variety of cellular pathways that are currently being
deciphered by academic and pharmaceutical laboratories for new target discovery. However,this field has been hampered by the lack of convenient assays suitable for discriminating the Sp
family members and performing high numbers of experiments.
To overcome this, Active Motif is introducing a high-throughput assay to quantify Sp1 and Sp3
activation17. TransAM Kits combine a fast and user-friendly ELISA format with a sensitive andspecific assay for transcription factors. TransAM Sp1 and Sp1/Sp3 Kits contain a 96-well plateon which has been immobilized an oligonucleotide that contains a Sp1 consensus binding site
(5´-GGGGCGGGG-3´). Sp1 and Sp3 contained in nuclear extract specifically binds to this oligonucleotide. The primary antibody used in TransAM Sp1 and Sp1/Sp3 Kits recognizes an accessible
epitope on Sp1 or Sp3 protein upon DNA binding. Addition of a secondary HRP-conjugatedantibody provides a sensitive colorimetric readout that is easily quantified by spectrophotometry.
Once the nuclear extracts are prepared, this assay is completed in less than 3.5 hours. As thisassay is performed in 96-well plates, a large number of samples can be handled simultaneously,
enabling high-throughput automation. This assay is specific for Sp1 and Sp3 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 Sp1 and Sp3 activation using as little as
0.6 µg of nuclear extract from MCF-7 cells. A comparable assay using EMSA required 2.5 µg of
nuclear extract and a 3-day autoradiography.
TransAM Sp1 and Sp1/Sp3 have many applications including the study of Sp1 transcriptional
activity regulation and protein structure/function studies of Sp1/Sp3 and their mutated variants inareas such as angiogenesis, metastasis, tumorigenesis and many more.

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4Kit Performance and Benefits
The TransAM Sp1 and Sp1/Sp3 Kits are for research use only. Not for use in diagnostic
procedures.
Detection limit: < 0.6 µg nuclear extract/well. TransAM Sp1/Sp3 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 Sp1/Sp3 detects Sp1 and Sp3 from human, mouse and rat origin.
Assay time: 3.5 hours. TransAM is 20-fold faster than EMSA.

Kit Components and Storage

Sp1/Sp3 activity (OD450 nm )10.75
Sp3 (treated extract)
Sp3 (untreated extract)

0.5

Sp1 (treated extract)
Sp1 (untreated extract)

0.25

0

0

2.5

5

7.5

10

Extract (µg/well)

Monitoring Sp1 and Sp3 activity with the TransAM Sp1/Sp3 Kit: Different amounts of untreated and H2O2post treated nuclear extracts from MCF-7 cells are tested for Sp1 and Sp3 activity by using the TransAM Sp1/Sp3 Kit. These curves are provided for demonstration only.

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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

Sp1 antibody
and/orSp3 antibody

11 µl / 55 µl-20°C for 6 months

11 µl / 55 µl-20°C for 6 months

Anti-rabbit HRP-conjugated IgG

11 µl / 55 µl(0.4 µg/µl)

4°C for 6 months

Wild-type oligonucleotide AM22100 µl / 500 µl(10 pmol/µl)-20°C for 6 months

Mutated oligonucleotide AM22100 µl / 500 µl(10 pmol/µl)-20°C for 6 months

MCF-7 nuclear extract

40 µl / 200 µl(2.5 µg/µl)-80°C for 6 monthDithiothreitol (DTT)

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

Protease Inhibitor Cocktail

100 µl / 500 µl-20°C for 6 months

Poly [d(I-C)]

100 µl / 500 µl(17 µg/ml)-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 month

Developing Solution

11 ml / 55 ml

4°C for 6 months

Stop Solution

11 ml / 55 ml

4°C for 6 months

96-well Sp1 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 ofreading at 450 nm (655 nm as optionalreference wavelength)• Detergent (NP-40)

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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.
Preparation of Complete Binding Buffer
Prepare the amount of Complete Binding Buffer required for the assay by adding 2 µl of DTT
and 10 µl of 17 µg/ml poly[d(l-C)] per ml of Binding Buffer AM1 (see the Quick Chart for Preparing
Buffers in this 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 form clumps, therefore homogenize the buffer by incubating at 50ºC for 2 minutes andmixing 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 to room temperature and vortexing for 1 minute prior to use. Dilute both primary and HRPconjugated secondary antibodies to 1:1000 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 MCF-7 nuclear extract is provided as a positive control for Sp1 and Sp3 activation. Sufficient
extract is supplied for 25 reactions per plate. This extract is optimized to give a strong signal
when used at 5 µg/well. We recommend aliquoting the extract in 10 µl fractions and storing at-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 Sp1 and Sp3 binding
in order to monitor the specificity of the assay. Used at 20 pmol/well, the oligonucleotide will
prevent Sp1 and Sp3 binding to the probe immobilized on the plate. Conversely, the mutatedconsensus oligonucleotide should have no effect on Sp1 and Sp3 binding. Prepare the required
amount of wild-type and/or mutated consensus oligonucleotide by adding 2 µl of appropriate
oligonucleotide to 43 µl of Complete Binding Buffer per well being used (see the Quick Chart for
Preparing Buffers in this section). To allow for optimum competition, add the oligonucleotide to
the well prior to addition of the cell extract.
Optional- Preparation of standard curve
For those who wish to quantify the amount of Sp1 in their samples, Active Motif offers recombinant Sp1 for use as a protein standard (see Appendix, Section B. Related Products).
1.

Begin with a 100 ng/µl working stock of recombinant protein (use the TransAM Complete Lysis
Buffer to dilute the protein). Set up a standard curve in duplicate using the following concentrations: 32, 16, 8, 4 and 0 ng/µl. Note: The preceding range is provided as guidance, abroader range of values may be used.

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8

2.

Make up a 32 ng/µl solution by adding 16 µl of the 100 ng/µl working stock to 34 µl ofComplete Lysis Buffer. Next, pipette 25 µl of Complete Lysis Buffer into the 3 remaining
tubes. Use the 32 ng/µl solution to prepare a dilution series as indicated below. Be sure tomix each tube thoroughly before each transfer. The 32 ng/µl standard serves as the highstandard, while Complete Lysis Buffer alone serves as the 0.0 ng/µl.

25 µl

32 ng/µl

25 µl

16 ng/µl

25 µl

4 ng/µl

8 ng/µl

3.
10 µl from each tube will be aliquoted to the wells in Step 1, No. 2 of the protocol
and will correspond to the following quantities of Sp1: 320, 160, 80, 40 and 0.0 ng/well.

Quick Chart for Preparing Buffers
1 well

1 strip(8 wells)

6 strips(48 wells)

12 strips(96 wells)

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

DTT
Poly [d(l-C)]
Binding Buffer AM1
TOTAL REQUIRED

0.09 µl0.45 µl44.5 µl
45 µl

0.7 µl
3.6 µl356 µl360 µl

4.3 µl21.6 µl2.134 ml
2.16 ml

8.6 µl43.2 µl
4.268 ml
4.32 ml

Complete Binding Buffer
with wild-type or mutatedoligonucleotide

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 m21.6 m
216 ml1X Antibody Binding Buffer*

Distilled water
10X Ab Binding Buffer AM3
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

Reagents to prepare

Components

Complete Lysis Buffer* Volumes listed refer to the preparation of buffer for diluting both the primary & secondary antibodies.

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9Sp1 & Sp1/Sp3 Transcription Factor Assays
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. Multichannel 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.
Step 1: Binding of Sp1 and/or Sp3 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 mutatedoligonucleotide (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 12.
Positive control wells: Add 5 µg of the provided MCF-7 nuclear extract diluted in 10 µl ofComplete Lysis Buffer per well (2 µl of extract in 8 µl of Complete Lysis Buffer per well).
Blank wells: Add 10 µl Complete Lysis Buffer only per well.
OPTIONAL – Protein standard wells: Add 10 µl of the appropriate protein standard dilutedin Complete Lysis Buffer to each well being used (see page 8, Preparation of standard
curve).

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 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.

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-rabbit 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).
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10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 2-5 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.

OPTIONAL – Calculation of results using the standard curve
If you have generated a standard curve using Active Motif’s recombinant Sp1 protein, average
the duplicate readings for each standard, control, and sample and subtract the optical density(OD) obtained from the zero standard.
Plot the OD for the standards against the quantity (ng/well) of the standards and draw the best
fit curve. The data can be linearized using log/log paper and regression analysis may also be
applied.
To quantify the amount of Sp1 in the samples, find the absorbance value for the samples on
the y-axis and extend a horizontal line to the standard curve. At the intersection point extend avertical line to the x-axis and read the corresponding standard value. Note: If the samples havebeen diluted, the value read from the standard curve must be multiplied by the dilution factor.
Example curve:
The following standard curve is provided for demonstration only. A standard curve should bemade every time an experiment is performed.
Sp1 (Baculovirus) Recombinant protein
Signal range 40-320 ng/well

Sp1 activation (OD450)1

y = 0.003x + 0.005 r2 = 0.975

0.75

0.5

0.25

0

0

100

200

300

Sp1 Recombinant Protein (ng)www.activemotif.com

11

400

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 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 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.
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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12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.

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13

References
1.
2.
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Kadonaga J.T., Courey A.J., Ladika J. and Tjian R. (1988) Science 242: 1566-70.
Suske G. (1999) Gene 238: 291-300.
Saffer J.D., Jackson S.P. and Annarella M.B. (1991) Mol. Cell. Biol. 11: 2189-99.
Marin M., Karis A., Visser P., Grosveld F. and Philipsen S. (1997) Cell 89: 619-28.
Kingsley C. and Winoto A. (1992) Mol. Cell. Biol. 12: 4251-61.
Pugh B.F. and Tjian R. (1990) Cell 61: 1187-97.
Perkins N.D., Agranoff A.B., Pascal E. and Nabel. G.J. (1994) Mol. Cell. Biol. 14: 6570-83.
Strom A.C., Forsberg M., Lillhager P. and Westin G. (1996) Nucleic Acids Res. 24: 1981-6.
Karlseder J., Rotheneder H. and Wintersberger E. (1996) Mol. Cell. Biol. 16: 1659-67.
Hata Y., Duh E., Zhang K., Robinson G.S. and Aiello L.P. (1998) J. Biol. Chem. 273:
19294-303.
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14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

Sp1 or Sp3 is poorly expressed orinactivated in nuclear fractions

Perform a time course for Sp1 or Sp3
expression in the studied cell line

Extracts are not from human,
mouse or rat origin

Perform study with a human, mouse or
rat model

15

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:877.222.9543
760.431.1263
760.431.1351
tech_service@activemotif.com

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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

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Direct:Fax:
E-mail:+81 (0)3 5225 3638
+81 (0)3 5261 8733
japantech@activemotif.com

Active Motif China
Direct: (86)-21-20926090
Cell Phone: 18521362870
E-mail: techchina@activemotif.com

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16