OHS026
Safe Work Procedure
Faculty/Division
Medicine
Document number
STGCL.SWP.41.1
School/ Divisional Unit
St George Clinical School
Initial Issue date
01/03/2010
Current version Current Version
1.0
Issue date: 01/03/2010
Next review date
01/03/2012
The Writing Safe Work Procedures Guideline (OHS027) should be consulted to assist in the completion of this form.
Safe Work Procedure Title and basic description
Title: Electrophoretic Gel Shift Assay (EMSA)
Description: Formation of complexes between protein and radioactively-labeled double stranded DNA probes
followed by electrophoresis through acrylamide gel
Associated risk assessment title and location: STGCL.RA.41.1 Risk assessment for Carrying out a Gel Shift Assay
Describe the activity or process
Electrophoretic mobility shift assays (EMSAs) are also known as mobility shift electrophoresis, gel shift assay, gel
mobility shift assay, band shift assay or gel retardation assay. A minor variant of the technique were antibodies are
used to interact with a specific protein in called a super-shift assay.
EMSA is a technique used to study nucleic acids and protein interactions; most commonly DNA:protein interactions.
This assay is used to determine if a protein (or mixture of proteins) contain an activity that recognizes a specific DNA
sequence.
The assay is carried out in vitro by means of electrophoresis in a polyacrylamide gel as shown schematically in Fig. 1
Wells
Direction
of
movement
Shifted
probe
Free
probe
1
2
Fig. 1. Schematic
3 representation of EMSA. Lane
1. Probe alone; Lane 2. Negative control; Lane 3.
Positive control. The wells and the direction of
movement are indicated
The DNA is labeled with 32P to facilitate detection (this is the probe). The probe is incubated with the purified protein
or mixture of proteins (eg, cell lysate or nuclear extract) in a binding buffer and then the mixture is loaded into the
wells of the gel and electrophoresed for 2 to 3 h. The gel is then dried in a gel dryer and exposed to an X-ray film for
24 to 48 and then developed. The free probe (ie not bound to DNA) migrates faster and appears at the bottom of the
gel (Fig.1, lanes 1 & 2). If the probe is bound by DNA is migrates slower through the gel due to changes in (shifted
probe, Fig. 1, lane 3).
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Safe Work Procedure
Date Effective: 01/01/2007
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Current Version: 1.2, 15/08/2007
Describe the activity or process
Procedure
A) Labeling of the oligonucleotide
1- Annealing of the oligonucleotide.

Prepare 5 ml of 10X ennealing buffer:
o
o
o
40 mM Tris, pH 7.5
20 mM MgCl2
50 mM NaCl

Add the following to a PCR tube:
o 5
o
o
o
Note: the final concentration of the double
Use the PCR machine to run the following program
o 96oC, 10 min
o 90oC, 5 sec
o 85oC, 5 sec
o 80oC, 10 sec
o 70oC, 10 sec
o 60oC, 10 sec
o 25oC, stop
NB: Alternatively it can be done on a hot block in 1.5 ml Eppendorf tubes:
heat for 5 min at 96oC on hotblock, switch off hot-block and allow to cool to room temperature slowly (about 2h, it can be
left overnight).
2- Labeling Reaction. NB: trained personnel MUST carry out this procedure in the Radiation Laboratory.
Ware the appropriate PPE, radiation badge and work behind a perpex screen.

o
o
o
o
o
Add the following to a 1.5 ml Eppendorf tube
2
32P]
Place the tube(s) in a perpex container and incubate at 37 oC for 1h in the incubator in the radiation room. Then
incubate the tubes at 68oC in a water bath for 10 min (risk of burns by hot water or steam, wear heat resistant gloves).
3- Removal of unincorporated radionucleotides using Amersham G50 spin columns
o
-50 column (Amersham
Biosciences) into 1.5ml microcentrifuge tube at 700 g for 1min (Eppendorf benchtop centrifuge).
o
Transfer column into a fresh 1.5ml microcentrifuge tube and load sample on top of dried beads of
column. Centrifuge at 700 g for 2 mins. Dispose of resin and other waste in the appropriate bin.
o
Estimate efficiency of labeling reaction using the
o
Dilute probes to a final activity of 2x105 cpm
o
Keep probes on ice if using that day, or store for longer periods at -20C.
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4- Preparation of polyacrylamide gel (5%)
Prepare 10X TBE buffer:
o 108g Tris
o 55g boric acid
o 7.4g EDTA (or 9.3g of Na2EDTA.2H2O), pH to 8.2 in 900 ml and make up to 1L with dH2O. Store at
room temperature
Prepare 0.5X TBE buffer
o 100 ml 10X TBE
o 1900 ml dH2O
Prepare Gel mixture
o 8.5 ml of 30% acrylamide (29:1) (Note: this is a hazardous substance. Use only in a chemical
fume hood)
o 2.5 ml 10 X TBE buffer
o 0.5 ml 10% APS
o 38.7 ml water
o 50
5- Preparation of sample for EMSA

Make 2X gel shift buffer (for 10 ml)
Component (stock solution)
Volume
1M Tris, pH 7.5
100% glycerol
2 ml
1M MgCl2
0.5M EDTA
1M KCl
1.2 ml
1M DTT*
l
10 mg/ml BSA*
dH2O
5.75 ml
Final concentration
50mM
20%
12mM
1mM
120mM
1mM
--
This buffer can be store at –20oC for up to 1 year.
Add just before use

Make loading dye (fro 5 ml)
Component (stock solution)
Volume
1M Tris, pH 7.5
solid sucrose
2g
solid bromophenol blue
0.01g
Final concentration
30 mM
40%
0.2%
Store in aliquots at –20oC for up to 1 year

o
o
o
o
o
o
o
5 cpm)
-dC)
dy (if required for supershift)
10 to 200X molar excess of cold probe (if required)
dH2
Incubate on ice for 30 min.

6- Running Samples on EMSA Gel
While incubating samples, pre-run gel in 0.5x TBE at 180V. The gel and buffer needs to be precooled to this temperature well before running. This can be done with a buffer chiller or by running
in the cold room.
o
o
Load samples, run gel at 240V, for approximately 3hrs (must be judged empirically)
o Dry gel onto 2 pieces of Whattman paper (use gel drier at 80C for 1h). Be careful not to
contaminate the dryer
Expose gel to x-ray film (Hypercassette, MS intensifying screen & Kodak MS film for extra sensitivity),
usually overnight.
o
Develop in the dark room
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List all resources required including plant,
chemicals, personal protective clothing and
equipment, etc
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Gloves
Lab coat
Safety glasses
Radiation monitor
Large protein gel electrophoresis tank
Protein electrophoresis power pack
Microcentrifuge
Perpex boxes
Fume hood
Heating blocks or PCR machine
Incubator
Water bath
Gel dryer
Whatman paper
32P-ATP
X-ray cassette and X-ray film
Tris (Amresco, 0497-1kg)
Ethylenediaminetetraacetic acid (EDTA)
KCl
Glycerol (Sigma, G-5516)
DL-Dithiothreitol (DTT) (Sigma, D5545)
MgCl2 (BDH, 10149)
Boric acid (Sigma, B7901)
30% acrylamide/bis 29:1 (BIO-RAD, 161-0156)
Ammonium persulfate (APS) (Sigma, A3678)
N,N,N′,N′-Tetramethylethylenediamine (TEMED) (Sigma, T9281)
Nonidet P40 (NP40)
Poly dI-dC
List potential hazards and risk controls including
specific precautions required
Mechanical/Electrical
Protein electrophoresis equipment: Electrical appliance – electrocution hazard
Heating Blocks: Electrical appliance – electrocution hazard. Hot, burn hazard.
Gel Dryer: Electrical appliance – electrocution hazard. Hot, burn hazard.
Microcentrifuge: Electrical appliance – electrocution hazard. Spinning rotor- physical injury hazard.
Radiation
32
P-ATP: Radiation Hazard- Radiation exposure, potentially carcinogenic and teratogenic
Chemical Hazards
Tris: Irritant
Ethylenediaminetetraacetic acid (EDTA): Toxic, irritant, combustible
DL-Dithiothreitol: Irritant
Boric acid: Irritant
30% acrylamide/bis 29:1: Toxic to peripheral and central nervous system, carcinogenic, mutagenic, developmental toxicity. Severe
exposure can cause death. Avoid ingestion, inhalation and skin exposure.
Ammonium persulfate (APS): Harmful if swallowed
N,N,N′,N′-Tetramethylethylenediamine (TEMED): Flammable
Risk controls
Standard PPE lab coat, goggles and gloves.
Electrical equipment testing
Specific Risk controls
Radiation: Radiation Protection Training, use of shielding, time and distance controls
Irritant and toxic chemicals –weigh out and make solutions in fume hood
Special attention to Acrylamide: Handle in fume hood, wear double gloves see below for spills
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Safe Work Procedure
Date Effective: 01/01/2007
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Current Version: 1.2, 15/08/2007
List emergency shutdown instructions
For electrical appliances, the emergency cutoff switch (red button) is located on the wall near the entrance
to the lab 3.13.
Acrylamide:
In case of contact, immediately flush skin with plenty of water for at least 15 minutes while removing contaminated
clothing and shoes. Cover the irritated skin with an emollient. Cold water may be used.Wash clothing before reuse.
Thoroughly clean shoes before reuse. Get medical attention immediately.
List clean up and waste disposal requirements
The radiation lab working areas should be check for contamination with radioactive material with the Geiger counter.
All contaminated material should be disposed of in the corresponding waste bins provided.
All solutions should be disposed of as chemical waste.
List legislation, standards and codes of practice used
in the development of the SWP
The NSW Radiation Control Act (1990) and Regulation (2003)
NSW OHS Act 2000
NSW OHS Regulation 2001
AS/NZS 2243.2:2006. Safety in laboratories. Part 2: Chemical aspects
AS/NZS 2243.3: 2006 Safety in laboratories Part 3: Microbiological aspects and containment facilities
AS/NZS 2243.6-1990. Safety in laboratories. Part 6: Mechanical Aspects.
AS/NZS 2243.3:2006 Safety in Laboratories Part 7 Electrical aspects
AS/NZS 2161.1:2000 Occupational Protective Gloves – Selection, Use and Maintenance
Supervisory approval, training, and review
Supervisor: Prof Beng Chong (Medicine)
Signature:
Supervisor:
Signature:
Supervisor:
Signature:
Supervisor:
Signature:
Plant custodian: Prof Beng Chong
Signature :
List competency required – qualifications, certificates, licencing, training - eg course or instruction:
Completion of Radiation Protection Training and EPA Radiation License
Training in this SWP
SWP review date: 01/03/2012
Responsibility for SWP review: Jose Perdomo
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Safe Work Procedure
Date Effective: 01/01/2007
Uncontrolled document when printed
Current Version: 1.2, 15/08/2007