EMSA Kits

DRAFT March 21, 2007 5:46 pm

EMSATtile.fm

EMSA "Gel Shift" Kit

User Manual

F, 2007 5:46 pmEMSATtile.fm

About the User Manual

Who Should Read this Manual

Anyone that has purchased an EMSA “Gel Shift” Kit from Panomics to measure the binding activity of a specific TF from nuclear extracts.

This manual provides the following:

♦ Kit contents and storage conditions

♦ Assay procedures

♦ Troubleshooting

Safety Warnings and Precautions

CAUTION All chemicals should be considered potentially hazardous. We recommend that this product and its components be handled by those trained
in laboratory techniques and be used according to the principles of good laboratory practice.

Note This product is intended for research use only. It is not for diagnosis of disease in humans or animals.

About the EMSA “Gel Shift” Kit

Introduction to EMSA Kits

Panomics’ Electrophoretic-Mobility Shift Assay (EMSA) Kits are useful tools for identifying proteins that interact with DNA. This rapid technique is
based on the separation of free DNA from protein/DNA complexes due to the differences in their electrophoretic mobility in native (non-denaturing)
polyacrylamide gels. When a protein binds specifically to a labeled dsDNA sequence, it migrates slower than non-bound dsDNA in a polyacrylamide gel,
thus resulting in discrete bands corresponding to the individual protein/DNA complex. A typical EMSA experiment is performed by incubating a
biotin-labeled transcription factor (TF) Probe with treated and untreated nuclear extracts. The protein/DNA complexes are separated on a
non-denaturing polyacrylamide gel. The gel is transferred to a nylon membrane and detected using strepatvidin-HRP and a chemiluminescent substrate.
The shifted bands corresponding to the protein/DNA complexes are visualized relative to the unbound dsDNA. The bands are visualized after
exposure to film or chemiluminescent-imaging system. For more specific binding, you may wish to add unlabeled specific dsDNA probe
(cold probe; provided) to the protein/DNA reaction mixture which competes with the labeled dsDNA probe (biotin-labeled probe) for binding to the
protein. This causes the labeled probe to migrate to the bottom of the gel and reduces the intensity of the shifted band.
Panomics’ EMSA Kits are suitable for validation of results obtained using Procarta™ Transcription Factor Assay, TranSignal™ Protein/DNA Arrays
(Cat. #s MA1010 to MA1015) or to validate binding activity of a specific transcription factor (protein) to DNA. Enough reagents are provided to perform
25 binding reactions. Panomics' TF EMSA Kits measure the activity of specific transcription factors in nuclear extracts. The assay is highly specific,
precise, and requires a minimum of 5–10 μg of protein/well. Panomics currently offers more than 400 EMSA assays.

Panomics’ EMSA Kit Contents and Storage Conditions

Kit Contents and Storage

The EMSA Kit contains the following components. Refer to the product insert for quantities and details of components supplied. If stored properly,
reagents have a shelf-life of 6 months.

Component	Description	Storage	Location
5X Binding Buffer	Aqueous buffered solution for TF binding (50 μl)	–20 °C	Box 1
Poly d(I-C)	Used in TF binding reactions to act as competitor to TF Probe (25 μl)	–20 °C	Box 1
Loading Dye	Dye for loading samples and monitoring electrophoresis and transfer (25 μl)	–20 °C	Box 1
2X Blocking Buffer	Aqueous buffer for blocking during detection (50 mL)	–20 °C	Box 1
Control Nuclear Extract	Nuclear Extract prepared from HeLa cell line for use with control probe and cold control probe (5 μl)	–20 °C	Box 1
Control Probe	Control reagent (10 μl)	–20 °C	Box 1
Cold (unlabeled) Control Probe	Control reagent (10 μl)	4 °C	Box 1
Distilled H20	Distilled, purified water (500 μl)	4 °C	Box 1
Solution I	Chemiluminescent detection reagent (1 ml)	4 °C	Box 2
Solution II	Chemiluminescent detection reagent (1 ml)	4 °C	Box 2
Solution III	Chemiluminescent detection reagent (8 ml)	4 °C	Box 2
Streptavdin-HRP Conjugate	Used for binding to biotinylaled oligo on membrane (100 μl)	4 °C	Box 2
10X Detection Buffer	Aqueous buffer for detection step (10 ml)	15–30 °C	Box 2
10X Wash Buffer	Aqueous buffer for washing after addition of streptavidin-HRP (30 ml)	15–30 °C	Box 2
TF Probe	TF-specific Probe, biotinylated	–20 °C	Plastic Pouch
Cold (unlabeled) TF Probe	TF-specific Probe for use in competition assay	–20 °C	Plastic Pouch

Required Equipment and Materials Not Provided

Reagents

Item

30% Acrylamide/Bis Solution

Glycerol

Ammonium Persulfate (APS)

TEMED

10X TBE Stock Solution (1.0 M Tris, 0.9M Boric Acid 0.01 M EDTA)

Nuclear Extraction Kit

Protein Determination Kit

Equipment

Item

Electrophoresis Unit

Electroblotting Device

Dry Oven

UV Cross Linker (optional)

Chemiluminescent Imaging System (optional if using X-Ray film)

Rotating shaker

Thermal cycler

Materials

Item

Blotting Paper

X-Ray Film (optional if using Chemiluminescent Imaging system)

Neutrally Charged Nylon Membrane, 0.45 μm

Guidelines for Assay Design and Analysis

Preparing Samples Protein concentration of sample inputs should be at least 0.5–2 μg/μl.

General Guidelines

♦ Read this user manual and all product inserts before performing the assay.

♦ Store all reagents at the recommended temperatures.

♦ Use Panomics’ Nuclear Extraction Kit for best results.

Assay Procedures

Before You Start

♦ Thaw Positive Control and sample nuclear extracts on ice.

♦ Thaw TF-Specific Probe and TF-Specific Cold Probe on ice.

Preparing Nuclear Extracts

We recommend using a commercially available kit, such as Panomics’ Nuclear

Extraction Kit (P/N AY2002). Please note that the provided control nuclear extract is a

positive control only when used with the supplied control probe.

♦ We recommend using a concentration of ~5–10 μg/μl of Nuclear Extract. If your

samples have a different concentration, adjust the amount of water accordingly.

♦ When measuring protein concentration, we also recommend using the DC

Protein assay from Bio-Rad (Lowry assay).

Forming TF-DNA Complexes

1 Prepare nuclearextract

2 For each nuclear extract sample, combine the following components into a sterile 0.5-ml microcentrifuge tube (PCR tube).

• 1.0 μl Nuclear Extract

• 1.0 μl Poly d(I-C) (1μg/μL)

• 2.0 μl of 5X Binding Buffer

• 5.0 μl nuclease-free water

Mix above reagents and incubate at RT for 5 minutes

3 Add 1.0 μl of TF Probe and proceed to step 6. Total volume should be 10 μl.

4 (OPTIONAL) If a competition assay is desired (see Figure1, Lane 4), follow steps 4

and 5.

• 1.0 μl Nuclear Extract

• 1.0 μl Poly d(I-C) (1μg/μL)

• 2.0 μl of 5X Binding Buffer

• 3.0 μl nuclease-free water

Mix and incubate 5 min at room temp

5 Add 2.0 μl Cold TF Probe to the above mixture and incubate for 5 minutes at RT.

Then add 1.0 μl of labeled TF Probe and proceed to Step 6.

6 Incubate samples at 15 °C for 30 min in a thermal cycler

Gel Preparation Non-Denaturing Gel

1 Prepare a 6.0% non denaturing polyacrylamide gel. Be sure to dilute solutions/buffers, as described in the Appendix. Mix the following components
into a sterile 50-ml centrifuge tube (add in order listed):

• 1 ml of 10X TBE

• 4 ml of 30% Acrylamide/Bis

• 625 μL of 80% Glycerol

• 14.375 ml of deionized, sterile water

• 300 μL of 10% APS

• 20 μL Temed

Total volume is 20 mL

2 Cast the gel as per standard protocol.

3 Pre-chill 0.5X TBE to 4 °C before running your gel.

4 Run gel in chilled 0.5X TBE for 10 min at 120V before loading samples into gel. Prior to loading your samples, flush the wells with a transfer pipet.

5 Mix samples with 1 μL of Loading Dye provided and load 10 μL of sample to each lane.

6 Run the gel at 4°C (in an ice bath or refrigerator) at 120V until the dye reaches 1 inch from the bottom of the gel (Approx. time: 50-55 min).

Transfer Standard “wet-transfer” electroblotting procedure

1 Presoak Pall Biodyne B nylon membrane in 0.5X TBE. Prepare four sheets of gel-sized Whatman 3MM paper (8 x 10 cm). Presoak two sheets of Whatman
3MM paper in 0.5X TBE.

IMPORTANT Please ensure that only Pall Biodyne B nylon membrane is used. Any other membrane may cause high backgrounds

2     After electrophoresis, carefully remove one glass from the gel. Cover with one sheet of dry Whatman 3MM paper and the gel will stick to the paper.
       Gently lift the Whatman paper and gel away from the glass plate and note the orientation of the gel. Add an additional Whatman paper and soak in
       0.5X TBE. Sandwich the gel with the pre-soaked Biodyne B membrane and two sheets of presoaked Whatman paper then add the fiber pads on both
       ends.

3 Place the sandwich in an electoblotting device and fill tank with 0.5X TBE. Transfer for 30 minutes at 300mA.

IMPORTANT Ensure that the sandwich is oriented properly. The Biodyne B membrane should be closest to the positive pole (red) and the polyacrylamide
gel should be closest to the negative pole (black).

Immobilization and Detection Crosslinking and Detection

1 After transfer, remove the membrane from the sandwich of Whatman paper and gel and place between two fresh sheets of Whatman 3MM paper.

IMPORTANT If transfer was successful, the loading dye should be slightly visible on the membrane.

2 The oligos on the membrane can be fixed by baking the membrane for 1 hour in a dry oven at 80°C. Alternatively, the oligos can be fixed using a
UV crosslinker for 3 min. If UV crosslinking, ensure that the side of the membrane that was closest to the gel is exposed to the UV light source.

Note At this point in the experiment, you can choose to continue or stop at this point. The membrane is stable for several months if stored
between Whatman paper and in the dark.

3 Transfer the membrane to a new container containing 20 ml of 1X Blocking Buffer. If more than 1 membrane was prepared, each membrane will need
its own container. Lids from a 200 μl pipette tip box can be used for 8 x 10 cm blots.

4 Block the membrane by incubating at room temperature with the 1X Blocking Buffer for 15 min with gentle shaking.

5 Remove 1 ml of the 1X Blocking Buffer from the blot container and place into a clean microcentrifuge tube. Add 20 μl of Streptavidin-HRP to the tube and
vortex for 10 seconds.

6 Transfer the diluted Streptavidin-HRP mixture to the container with the blot and continue shaking at room temperature for another 15 min.

IMPORTANT When adding the mixture to the container, avoid pouring the contents of the microcentrifuge tube directly onto the membrane

7 Decant the diluted Steptavidin-HRP solution. Wash each membrane for 8 minutes, 3 times at room temperature with 20 ml of 1X Wash Buffer.

8 Add 20 ml of 1X Detection Buffer to each membrane and incubate at room temperature for 5 min.

9 Take a plastic sheet protector and cut the sheet protector so that the membrane fits between the inside of the pocket of the two sheets.
Alternatively two pieces of transparency film can be cut and the membrane sandwiched between each piece.

10 Prepare 2 ml of working Substrate Solution for each membrane by mixing (in order): 200 μl Solution I with 200 μl Solution II, briefly vortex,
then add 1.6 ml of Solution III and mix.

11     On a flat and even surface, remove the top plastic sheet of the “sandwiched” membrane and pipette 2 ml of the mixed working Substrate Solution onto
          each membrane. Replace the top plastic sheet and ensure that the substrate solution is evenly distributed over the membrane with no air bubbles.
          Incubate at roomtemperature for 5 min.

IMPORTANT Remove excess substrate by gently applying pressure over the top sheet and using a paper towel to wipe up any excess fluid.

12 Expose the membranes using either Hyperfilm ECL (2-10 minutes) or a chemiluminescent imaging system (12-15 min), such as the FluorChem Imager
from Alpha Innotech Corp.

IMPORTANT Several different exposure times to film or the imaging system may be needed for an optimal EMSA image.

Troubleshooting

Possible Problems and Recommended Solutions

Observation Possible Cause Recommended Action

Weak or no signal Insufficient Transfer Follow instructions in theuser manual for performing the assay

Exposure Time to Short Increase time of exposure

Membrane is too dry
Keep membrane moist during detection

Insufficient protein Confirm the concentration of the protein assay using a Lowry or Bradford assay. If needed, increase the amount of protein in the nuclear extract. We recommend preparing nuclearextracts with Panomics’ Nuclear Extraction Kit (P/N AY2002).

Target protein not activated (induced) Review induction procedures. You may need to change cell lines,inducer, or induction conditions

High background Samples overdeveloped Shorten the development time.

Non-specific protein bound to membrane Increase blocking or washing times

Wrong type of membrane used Only use Biodyne B, a neutrally charged nylon membrane

No Shift Observed The amount of extract will vary according to the extract preparation, DNAbinding affinity of the protein, and quality of the extract. Typically, 1-20 μg of extract should give a sufficient band shift result

There is no clear band in the lane Disassociation of protein and probe has occurred during running of the gel Keep all buffers and other components chilled while running the gelto minimize disassociation

There are multiple bands in the lane Some non-specific binding of probes can occur Determine specific bands by performing competition reactions with supplied unlabeled probes.

Appendix

♦ 100 ml or 1X Blocking Buffer: To 50 ml of deionized water, add 50 ml of 2X

Blocking Buffer. Mix well and store at 4°C

♦ 300ml of 1X Wash Buffer: To 270 ml of deionized water, add 30 ml of 10X Wash

Buffer. Mix Well and store at room temperature

♦ 100ml of 1X Detection Buffer: To 90 ml of deionized water, add 10 ml of 10X

Detection Buffer (provided). Mix well and store at room temperature.