Gelshift™
Chemiluminescent EMSA Kit(version A2)
Catalog No. 37341

Copyright 2010 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.
© 2010 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
Kit Components and Storage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Additional Materials Required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Protocols
Gelshift Chemiluminescent EMSA Protocol. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Appendix
Section A. Preparation of Nuclear Extract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Section B. Troubleshooting Guide. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Technical Services . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .14

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Overview
Electrophoretic mobility shift assays (EMSA), also known as gel shifts, gel retardation assays ormobility assays can be used to study DNA-protein interactions1-3. The principle behind EMSA relies on the fact that DNA-protein complexes migrate slower than DNA alone in a native polyacrylamide or agarose gel. This difference in electrophoretic separation of DNA-protein complexes
can be visualized as a “shift” in migration of the labeled DNA band.
The Gelshift™ Chemiluminescent EMSA Kit provides a non-radioactive method to detect DNAprotein interactions. In this method, cell extracts or purified factor are incubated with a biotin 3’
or 5’ end-labeled DNA probe containing the consensus binding site of interest. Samples are thenresolved by electrophoresis on a native polyacrylamide gel and transferred to a nylon membrane.
The biotin end-labeled DNA probe is detected using streptavidin conjugated to horseradish peroxidase (HRP) and a chemiluminescent substrate. The assay can be complete in approximately 5
hours.

product

formatcatalog no.

Gelshift™ Chemiluminescent EMSA

100 rxns

37341

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Kit Components and Storage
Gelshift Chemiluminescent EMSA Kits are for research use only. Not for use in diagnostic procedures. 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.
Reagents

Quantity

Storage

10X Binding Buffer

1 ml

-20°C

Biotin Control DNA

50 µl

-20°C

Unlabeled Control DNA

50 µl

-20°C

Control Nuclear Extract

125 µl-20°C

Poly d(I-C)

125 µl

-20°C

50% Glycerol

500 µl-20°C

1% NP-40

500 µl-20°C

1M KCl

1 ml

-20°C

100 mM MgCl2500 µl-20°C

200 mM EDTA, pH 8.0

500 µl

-20°C

1 ml

-20°C

Streptavidin HRP-conjugate

1.5 ml4°C

Chemiluminescent Reagent

80 ml

4°C

Reaction Buffer

80 ml

4°C

Blocking Buffer

500 ml4°C

4X Wash Buffer

500 ml4°C

Substrate Equilibration Buffer

500 ml4°C

5X Loading Bufferwww.activemotif.com

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Additional materials required• Biotin 3’ or 5’ end-labeled DNA target. Do not use DNA probes with internal biotin labels as
the internal label may inhibit the protein binding to DNA.
• Unlabeled DNA target for use in competition experiments to verify specificity of the DNAprotein complex• Nuclear extracts containing the target protein of interest. (Nuclear extracts can be preparedusing Active Motif’s Nuclear Extract Kit Catalog Nos. 40010 & 40410)• Polyacrylamide gel in 0.5X TBE (most systems use a 4-6% polyacrylamide gel in 0.5X TBE)
Commercially available gels such as Life Technologies 6% DNA Retardation Gel 1.0 mm,
10 well (Cat # EC6365BOX) can also be used
• Electrophoresis apparatus• Positively charged nylon membrane• Electroblotter or capillary transfer apparatus• High quality blotting paper• Clean sponges for electrophoretic transfer• Circulating waterbath• Plastic forceps• Cross-linking device such as a UV lamp or commercial cross-linking device with 254 nm
bulbs or a transilluminator equipped with 312 nm bulbs• Clean plastic trays or weigh boats for detection• Orbital shaker• X-ray film or CCD camera

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Gelshift Chemiluminescent EMSA Protocol
Read the entire protocol before use.
The Gelshift Chemiluminescent EMSA kit includes biotin-labeled and unlabeled control DNA as
well as a control nuclear extract. If the kit is being used for the first time, we recommend to run
the control system reactions to verify the kit components and procedure are working properly.
The control samples are from the Epstein-Barr Nuclear Antigen (EBNA) system. A description ofcontrol reactions and expected results are shown in Step 1.
Do not thaw the Control Nuclear Extract provided in the kit until immediately before use. Thaw
the remaining binding reaction components and place them on ice. Take care to avoid excessive
warming of DNA probes.
Biotin 5’ or 3’ end-labeled target DNA and unlabeled target DNA can be ordered directly fromyour oligo supplier. Internal biotin labels, such as those that arise from random prime labelingmethods, are not recommended because the internal biotin labels may inhibit binding of theprotein to DNA. A typical target duplex DNA is 20-35 bp for use in EMSA. The actual protein:DNA
binding sequence is frequently 10-15 bp. Longer probes can be used if the binding sequence isunknown or if multiple regulatory regions are being studied.
Sample nuclear extracts can be prepared using Active Motif’s Nuclear Extract Kit (Catalog Nos.
40010 & 40410). The amount of protein required for the binding reactions depends on theamount of active protein in the sample. If the protein being studied is abundant, 0.25 µg of lysate
may be sufficient for each binding reaction. However, if the protein is less abundant we recommend using 10 µg or more. Using a large excess of extract may lead to high background signal
and nonspecific bands. Some optimization may be required to determine the best conditions foryour sample system. The final volume of the binding reaction is 20 µl. If a large volume of nuclear
extract is required, remove excess salts in the extract by dialyzing into a buffer containing 200
mM salt before using in the Gelshift Chemiluminescent EMSA Kit.
This kit has been optimized for use with polyacrylamide mini (8 x 8 x 0.1 cm) gels. Please adjustelectrophoresis conditions and detection reagent volumes if using larger gels.

Step 1: Plan the Experiment1.

Control DNA and Control Nuclear Extract are included in the Gelshift Chemiluminescent
EMSA. It is recommended to perform a set of three control reactions as described in Step 3
each time an EMSA is performed.

2.

It is also recommended to perform a set of three binding reactions for each sample system
to verify the appropriate shift and protein:DNA specificity of the interaction. Optimization of
the sample system can be achieved by adding other components supplied in the kit such as
KCl4,5, glycerol, MgCl24,6 and detergent7,8 to determine their effects on the shift. Please read
Step 4 to determine the binding reactions required for the sample system.

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4Step 2: Prepare and Pre-Run Gel
1.

Prepare a native polyacrylamide gel in 0.5X TBE or use a pre-cast DNA retardation gel. The
appropriate polyacrylamide percent depends on the size of the target DNA and the binding
protein. Most systems use a 4-6% polyacrylamide gel in 0.5X TBE.

2.

Add 0.5X TBE to fill the electrophoresis unit to just below the bottom of the wells. Flusheach well with 0.5X TBE. Pre-run the gel for 30-60 minutes at 100 V for an 8 x 8 x 0.1 cm gel.

3.

During this pre-run of the gel, proceed to Step 3.

Step 3: Control DNA Binding Reactions
1.

Perform a set of three control reactions each time an EMSA is performed.
ReactionContents

Expected Result#1

Biotin Control DNA

No shift. Without the control nuclear extract,
the reaction lacks protein to bind DNA andcause a shift.

#2

Biotin Control DNA +Control Nuclear Extract

DNA shift. The target protein present in thecontrol nuclear extract will bind to the biotinlabeled control DNA and cause a shift ascompared to reaction #1.

#3

Biotin Control DNA +Control Nuclear Extract +Unlabeled Control DNA

No shift. The excess of unlabeled control DNA
competes for binding of the target protein in
the control nuclear extract. This verifies thesignal observed in reaction #2 is the result ofspecific DNA-protein interaction.

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2.

To set up the control reactions, add components in the order shown below to each tube.
Reagent
Ultrapure Water
10X Binding Buffer
50% Glycerol
100 mM MgCl2
1 µg/µl Poly d(I-C)
1% NP-40
Unlabeled Control DNA
Control Nuclear Extract
Biotin Control DNA
Total Volume

Final Concentration

Reaction #1

Reaction #2

Reaction #3

–
1X
2.5%
5 mM
50 ng/µl
0.05%
4 pmol1 unit20 fmol
–

12 µl
2 µl
1 µl
1 µl
1 µl
1 µl
–
–
2 µl
20 µl

11 µl
2 µl
1 µl
1 µl
1 µl
1 µl
–
1 µl
2 µl
20 µl

9 µl
2 µl
1 µl
1 µl
1 µl
1 µl
2 µl
1 µl
2 µl
20 µl

3.

Incubate control binding reactions at room temperature for 20 minutes.

4.

Add 5 µl of 5X Loading Buffer to each 20 µl binding reaction. Pipette up and down several
times to mix, do not vortex or mix vigorously.

4.

To set up Sample DNA Binding Reactions Proceed to Step 4, otherwise proceed to Step 5 onpage 7 and continue with the protocol for electrophoresis, transfer, cross-linking and detection of control samples.

Step 4: Sample DNA Binding Reactions
1.

Perform a set of three reactions for each sample system to verify the appropriate shift andprotein:DNA specificity of the interaction. (See Step 3.1 of Control DNA Binding Reactions formore details)

2.

If specific binding conditions are not already known, it is recommended to use minimal
reaction components. To further optimize sample binding, the addition of optional reagents
such as glycerol, detergent, KCl, MgCl2 or EDTA can be included in the binding reactions.
Bovine serum albumin and basic peptides have also been shown to enhance some DNAprotein interactions8-10.
Poly d(I-C) is provided as a nonspecific competitor DNA. This is suitable for most sample
systems, but if the target DNA sequence is GC-rich, try Poly d(A-T), salmon sperm or E.coli
DNA.
Nuclear protein extracts can be prepared using Active Motif’s Nuclear Extract Kit (Catalog
Nos. 40010 & 40410)

3.

Add components in the order shown below to each tube. To overcome strong nonspecificinteractions, a short pre-incubation may be required before adding the biotin-labeled
sample DNA.

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Reagent

Final Conc.

Reaction #1

Reaction #2 Reaction #3

Ultrapure Water–

10X Binding Buffer

1X

2 µl

2 µl

2 µl

1 µg/µl Poly d(I-C)

50 ng/µl

1 µl

1 µl

1 µl

4 pmol––Optional: 50% Glycerol*
Optional: 1% NP-40
Optional: 1 M KCl
Optional: 100 mM MgCl2
Optional: 200 mM EDTA
Unlabeled Sample DNA
Nuclear Extract–

Biotin-labeled Sample DNA

20 fmol

Total Volume

–

20 µl

20 µl

20 µl

* Too much glycerol in the binding reaction may cause vertical streaks along the edges ofthe lanes.
4.

Incubate sample binding reactions at room temperature for 20 minutes.

5.

Add 5 µl of 5X Loading Buffer to each 20 µl binding reaction. Pipette up and down several
times to mix, do not vortex or mix vigorously.

Step 5: Electrophoresis of Binding Reactions
1.

Once the pre-run of the polyacrylamide gel is finished (Step 2), turn off current to the unit.

2.

Flush each well with 0.5X TBE buffer. Then load 20 µl of sample containing loading buffer
onto the polyacrylamide gel.

3.

Run the gel at 100 V (for 8 x 8x 0.1 cm gel) until the bromophenol blue dye has migrated
approximately 2/3 to 3/4 down the length of the gel. The free Biotin Control DNA duplex
migrates just behind the bromophenol blue when using a 6% gel.

Step 6: Transfer of Binding Reactions to Nylon Membrane
1.

Soak a nylon membrane in 0.5X TBE buffer for a minimum of 10 minutes.

2.

Follow the manufacturer’s instructions for your electrophoretic transfer unit. Use a cleantransfer unit and clean transfer sponges. Avoid using sponges that have been used in Western blots. Use 0.5X TBE cooled to approximately 10°C with a circulating waterbath for thetransfer. Handle the nylon membrane only at the corners using clean forceps and powderfree gloves.

3.

Transfer at 380 mA (~100 V) for 30 minutes for mini gels (8 x 8 x 0.1 cm). Typical transfer
times are 30-60 minutes at 380 mA using a standard tank apparatus.
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4.

Once the transfer is complete, place the membrane with the bromophenol blue side up ona dry paper towel. The gel should not contain any visible dye if the transfer was complete.
Allow buffer on the nylon membrane surface to absorb into the membrane (~ 1 minute), but
do not let the membrane dry. Proceed immediately to Step 7.

Step 7: Cross-link Transferred DNA to Membrane
1.

There are three methods to choose from for cross-linking the DNA to the membrane.
Commercial UV-light cross-linker: Cross-link at 120 mJ/cm2 using a commercial UV-lightcross-linker instrument equipped with 254 nm bulbs (45-60 second exposure using the autocross-link function).
Hand-held UV lamp: Cross-link at a distance of approximately 0.5 cm from the membrane
for 5-10 minutes with a hand-held UV lamp equipped with 254 nm bulbs.
Transilluminator: Cross-link for 10-15 minutes with the membrane face down on atransilluminator equipped with 312 nm bulbs.

2.

After cross-linking, the membrane may be stored dry at room temperature for several daysor else immediately detected in Step 8. If the membrane is stored, do not allow it to get wet
until ready to proceed with Step 8.

Step 8: Detect Biotin-labeled DNA by ChemiluminescenceThis kit has been optimized for use with polyacrylamide mini (8 x 8 x 0.1 cm) gels. Please adjustelectrophoresis conditions and detection reagent volumes if using larger gels. Perform all blocking and detection incubations in clean trays or in plastic weigh boats on an orbital shaker.
Preparation of Blocking Buffer
Warm the Blocking Buffer in a 37-50°C water bath until all particulate is dissolved. Once the particulate is completely dissolved. The buffer may be used between room temperature and 50°C as
long as all the particulates remain in solution
Preparation of Streptavidin Conjugate/Blocking Buffer Solution
Prepare a 1:300 dilution of streptavidin conjugate/blocking buffer solution by adding 66.7 µl
Streptavidin-HRP conjugate to 20 ml Blocking Buffer. Mix prior to use.
Note:

The streptavidin conjugate/blocking buffer solution has been optimized to workat a 1:300 dilution and should not be modified.

Preparation of 1X Wash Buffer
Warm the 4X Wash Buffer in a 37-50°C water bath until all particulate is dissolved. Once theparticulate is completely dissolved, prepare the amount of 1X Wash Buffer required for the assay
as follows: Dilute 40 ml 4X Wash Buffer with 120 ml ultrapure water and mix prior to use.

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1.

Prepare the Blocking Buffer, Streptavidin Conjugate/Blocking Buffer Solution and 1X Wash
Buffer as stated above.

2.

Take the cross-linked membrane from Step 7 and place in a clean tray or plastic weightboat. Add 20 ml Blocking Buffer and incubate for 15 minutes at room temperature withgentle shaking on an orbital shaker.

3.

Decant the blocking buffer from the membrane and replace it with the Streptavidin Conjugate/Blocking Buffer solution. Incubate for 15 minutes at room temperature with gentle
shaking on an orbital shaker.

4.

Transfer the membrane to a new container and rinse briefly with 20 ml 1X Wash Buffer.

5.

Wash the membrane four times for 5 minutes each in 20 ml 1X Wash Buffer with gentle
shaking on an orbital shaker.

6.

Transfer the membrane to a new container and add 30 ml of Substrate Equilibration Buffer.
Incubate for 5 minutes with gentle shaking on an orbital shaker.

7.

Prepare Chemiluminescent Working solution by adding 6 ml of Chemiluminescent Reagent
to 6 ml Reaction Buffer. The Chemiluminescent Working Solution is light sensitive, therefore
we recommend keeping this working solution in an amber bottle and avoiding direct exposure to intense light. Short-term exposure to laboratory lighting will not affect the working
solution.

8.

Remove the membrane from the Substrate Equilibration Buffer, carefully blotting an edgeof the membrane on a paper towel to remove excess buffer. Place the membrane in aclean container or onto a clean sheet of plastic wrap. Make sure the membrane is on a flat
surface.

9.

Add the Chemiluminescent Working Solution to the membrane making sure the entire
surface of the membrane is covered. Alternatively, the membrane can be placed DNA side
down into a puddle of the Chemiluminescent Working Solution. Incubate the membranewith the substrate for 5 minutes at room temperature without shaking.

10. Remove the membrane from the substrate solution, carefully blotting an edge of the membrane on a paper towel to remove excess buffer. Do not allow the membrane to become
dry.
11.

Wrap the moist membrane in plastic wrap. Avoid creating bubbles or wrinkles with theplastic.

12. Expose the membrane to an appropriately equipped CCD camera, or place the membrane
in a film cassette and expose to X-ray film for 2-5 minutes. Develop the film according to the
manufacturer’s instructions. Exposure time may be adjusted to obtain the desired signal.

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9References
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.

Fried, M. and Crothers, D.M. (1981) Nucl. Acids Res. 9: 6505-6525.
Revzin, A. (1989) BioTechniques 7: 346-354.
Hendrickson, W. (1985) BioTechniques 3: 198-207.
Winston, R.L. et al. (1999) Biochemistry 38: 5138-5146.
Triplett, B. (1992) BioTechniques 13: 354-355.
Szczelkun, M.D. and Connolly, B.A. (1995) Biochemistry 34: 10724-10733.
Hodgson, J. and Enrietto, P.J. (1995) J. Virol. 69: 1971-1979.
Zhang, X.Y. et al. (1992) Anal. Biochem. 201: 366-374.
Kozmik, Z. et al. (1990) Nucl. Acids Res. 18: 2198.
Bannister, A. and Kouzarides, T. (1992) Nucl. Acids Res. 20: 3523.

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Appendix
Section A. Preparation of Nuclear Extract
For your convenience, Active Motif offers a Nuclear Extract Kit (Cat. Nos. 40010 & 40410). 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. Do not use Trypsin toremove cells as it may alter transcription factor activation states.

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.
Check the cells under a microscope to monitor the cell lysis. The cell membrane should becompletely lysed, while the nuclear membrane remains intact.

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. Discard the debris pellet.

9.

Determine the protein concentration of the extract by using a Bradford-based assay. It is
recommended to perform a 1:50 dilution of your samples for protein determination. A blanksample consisting of Complete Lysis Buffer diluted 1:50 should be run as control. Standard
controls should also be generated in Complete Lysis Buffer diluted 1:50.

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11Preparation of Buffers for Nuclear Extract
10X PBS
0.1 M phosphate buffer, pH 7.5

For 250 ml, mix:
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

0 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

Used sample DNA
without a biotin label

Make sure the sample DNA for the target of interest is 5’ or 3’
end-labeled with biotin

Not enough biotin
sample DNA used

Increase the amount of biotin sample DNA used per reaction

Sample DNA isdegraded

Verify the integrity of the sample DNA by spectrophotometry, orvisualization on an agarose gel

Poor transfer to nylon
membrane

Follow the transfer protocol and manufacturer’s instructions foryour transfer unit. If an agarose gel is used, a capillary transfer
may perform better. For polyacrylamide gels either capillary orelectrical transfers can be used.

Wrong membrane used

Use a positively charged nylon membrane

Blot dried out during
the detection steps

Make sure the solutions cover the entire membrane during
detection and the membrane stays moist

Poor cross-linking or
no cross-link

Check the efficiency of the cross-linker or ensure that the correct wavelength bulb was used for the method of cross-linking

4X Wash Buffer was not
diluted to 1X

Dilute the 4X Wash buffer to 1X in ultrapure water

Insufficient film
exposure

Increase the exposure time

Air bubbles

Eliminate air bubbles between the gel and the membrane duringtransfer. Also eliminate air bubbles when placing the membranein plastic for detection

Precipitate in the HRP
conjugate

Filter the conjugate through a 0.2 µm filter or centrifuge for 1
minute at maximum speed to pellet the precipitate

Transfer unit orsponges were dirty

Use clean equipment for the transfer. Use sponges that had notpreviously been used for Western blotting

Particulate in Blocking
or Wash Buffer

Follow the recommendations to warm the solutions to 37-50°C
to ensure particulate has dissolved. Do not dilute 4X Wash Buffer until particulate is completely dissolved

System not optimized

Try to optimize the system using the additional reagents provided: glycerol, KCl, MgCl2, NP-40, EDTA

Extract degraded

Do not thaw Control Nuclear Extract until immediately before
use. Use Active Motif’s Nuclear Extract Kit to prepare samples or
include protease inhibitors during sample preparation

Not enough extract

If the protein being studied is abundant, 0.25 µg of extract may
be sufficient. For less abundant proteins, use 10 µg or more.
Some optimization may be required for your sample system

Disrupted protein:DNA
complex by vortexing,
mixing or heating

Follow the protocol for the binding reactions. Try running the gelwith cold buffer

Did not use nonspecificcompetitor DNA

Include poly d(I-C), poly d(A-T), salmon sperm or E.coli DNA as anonspecific competitor. Add a short pre-incubation time before
adding the biotin-labeled sample DNA to the binding reactions

Spots or specklingHigh backgroundNo shift detected

All DNA shifted

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13

Technical Services
If you need assistance at any time, please call or send an e-mail to Active Motif Technical Service
at one of the locations listed below.

Active Motif North America
Toll free: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

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

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