OHS026
Safe Work Procedure
Faculty/Division
Medicine
Document number
SOMS.CGM.SWP057
Initial Issue date
26.6.09
School/ Divisional Unit
School of Medical Sciences, Oncology Research
Unit
Current version
Current Version
Next review date
1.0
26.6.12
Issue date
26.6.09
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: Western Blotting – Gel Running and transfer
Description: Procedure for measuring protein quantities by running samples through a polyacrylamide gel, then transferring these
proteins to PVDF membranes.
Associated risk assessment title and location: 57_RA_Western Blotting – Gel running and Transfer_NC
Describe the activity or process
Western Blotting (please note: the preparation of acylamide solutions and pouring of acrylamide gels are described in the SWPs
“Prepartion of acrylamide solutions” and “Pouring of acrylamide gels” respectively. The Antibody probing of membranes is described in
the “Western Blotting- Membrane Probing” SWP.
Western blotting is here the process of applying a current to a poly-acrylamide gel (see “Pouring of Acrylamide gels” SWP), which is
immersed in a liquid buffer filled tank. This tank is connected to a power converter which provides electrical current to diodes within the
tank.The gel is loaded with samples which will migrate through the gel, according to their molecular weight, upon current
electrophoresis. This gel is then placed adjacent to a poly-vinyl membrane, and through a separate tank and electrical current protocol,
those proteins which are dispersed throughout the gel are transferred to the poly-vinyl membrane. This membrane is then stained, and
probed with a specific antibody or antibodies to detect proteins of interest (see “Western Blotting- Membrane Probing” SWP).
1. Gel Loading
Take set gel (See “Pouring Polacrylamide Gels” SWP) and load into gel running cassette. If only one
gel is being run, load a buffer dam in the opposing slot to the gel. Slide cassette into running tank. Pour
in ~800ml SDS Running buffer in main chamber, and ~100ml SDS running buffer into cavity between
gel, cassette and buffer dam. Pipette in appropriate volumes (5 – 25ul) of sample in wells within the gel.
When finished, affix the lid to the tank, ensuring that the cathodes and anodes are aligned (ie red
electrode on the lid to red electrode on the tank, black to black).
2. Gel Running
Plug the tank leads (again red to red and black to black) into the BIO-RAD powerpac. Turn the power
pac on. Set the powerpac to Volts constant, and adjust the voltage to between 70 – 100V. Hit the
“running man” button to initiate current through the gel apparatus. Once gel has finished running
(between 10mins and 2 hours later, depending the size of proteins to be resolved), hit the “flat hand”
button to stop the current. Turn the powerpac off. Unplug the running tank, and place it in the sink.
Remove the gel cassette from within, and place on absorbent material on the adjacent bench. Pour the
running buffer from the upper chamber into the main chamber. Into a large beaker, pour the used
running buffer. This buffer can be kept in a bottle for reuse, or discarded in the dedicated 20L drum kept
in the laboratory.
3. Gel removal
Open the gel cassette and remove the gel and buffer dam. Using a “gel releaser” (rectangular plastic
device provided by BIO-RAD), pry the two pieces of glass encasing the gel apart. NOTE: Do NOT use
forceps to separate these glass plates, as plates will break.
4. Assembling the Gel transfer to PVDF membrane apparatus:
Open the gel transfer cassette, and place the black surface down into the cassette well of the transfer
tray. Fill the tray halfway with transfer buffer. Thoroughly wet two foam pads in the transfer buffer, and
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Safe Work Procedure
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place one on top of the black (now bottom) surface of the transfer cassette. Wet one piece of Whatman
paper (cut to be smaller than the foam pad but larger than the gel) and place on top of the foam pad.
Wet the “gel releaser” in transfer buffer, and use it to lift the gel off the (now single) glass plate. Place
the gel squarely onto the wet piece of Whatman paper, which is directly on the foam pad, which is
directly on the black surface of the cassette. Pour ~40ml Methanol into a small blue western blot
rectangular container. Take one cut piece of PVDF and, using flat forceps, immerse it in the methanol
for 10 seconds, and then into the transfer buffer in the tray (Methanol can be stored for resuse in sealing
future membranes). Place the membrane so that it adheres entirely to the gel. Using a clean, transfer
buffer wet 15ml tube, roll out any air bubbles between the gel and the membrane. Wet another cut piece
of Whatman paper in transfer buffer, and lay this over the membrane. Again roll out any air bubbles with
the 15ml tube. Thoroughly wet another foam pad, and place this on to the now layered Black plasticWhatman paper-Gel-Membrane-Whatman paper sandwich (see Figure 1.). Close the transfer cassette
and place into the transfer tank, being careful to align the black side of the cassette with the black side
of the transfer tank. Fill the tank with Transfer Buffer (~1.2L), and place a solid ice pack into the transfer
tank. Fill a large eski ¼ full with ice, and form a well in the ice large enough for the tank. Place the tank
into this well, plug the tank lid on, ensuring the black goes to the black, and plug the lid into the
powerpac.
5. Transferring the gel to PVDF membrane
Turn the powerpac on. Set the volts constant to 80V, and put the timer on for 120 mins. Hit the “running
man” button to start the transfer. 120 mins later when the transfer is finished, turn the power pac off,
and remove the tank lid. Put a large plastic beaker in the sink, and pour the used transfer buffer into it.
This buffer can then be decanted into a bottle for reuse, or can be disposed of in the dedicated 20L
waste
drum in the lab. Remove the Transfer cassette from the tank, and place on absorbent material on the
bench. Open the cassette, and carefully remove the top foam pad (furthest from the black side), and the
Whatman paper nearest it. Using flat forceps, remove the membrane adjacent to the gel, and place into
a clean blue rectangular container for western blotting. Cover the membrane with distilled water. If the
gel is to be stained, place in a dedicated plastic container with a lid. Always discard of polyacrylamide
gels in the purple cytotoxic containers. Discard the whatman paper in the yellow biohazard bins. Wash
the tank, cassette, and foam pads with distilled water and leave to dry. Dispose of eski ice from transfer
down the sink.
Figure 1. The order of assembling the gel to membrane transfer cassette.
Grey/Clear (Red)
Foam Pad
Whatman Paper
Membrane
Gel
Whatman Paper
Foam Pad
Black (Black)
List all resources required including plant,
chemicals, personal protective clothing and
equipment, etc
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List potential hazards and risk controls including
specific precautions required
Acrylamide – Toxic, carcinogen/mutagen, neurotoxin - may cause cancer and heritable genetic damage. Toxic in
contact with skin and if swallowed. Danger of serious damage to health by prolonged exposure through inhalation, in
contact with skin or if swallowed. May cause eye irritation and allergic skin reaction. Polymerization may occur from
excessive heat or contamination. Wear safety goggles where splashing is possible. Wear enclosed shoes, nitrile gloves,
and gown.
Methanol – Toxic, irritant. Keep away from naked flames and ignition sources. Methanol used for the preparation of
Transfer Buffer is decanted within the laboratory. Methanol for storage within the laboratory is decanted within the
fume hood. Methanol for sealing membranes is kept in small working volumes (<50ml) on the bench. Wear enclosed
shoes, gloves, and gown. Wear goggles when a splash risk is possible.
Bis-acrylamide (Bis-Acrylagel) – Irritant. Irritating to eyes, respiratory system and skin. Harmful if swallowed,
inhaled or absorbed. Toxic if ingested. Wear safety goggles where splashing is possible. Wear enclosed shoes, gloves
and gown.
TEMED - Corrosive. May flame on contact with heat or fire. Moderately toxic via oral route. Continuous exposure of
low levels of vapour can cause a blurring of vision called "Blue Haze". Wear gloves, mask, goggles, and gown. Keep
away from ignition sources.
Ammonium Persulfate (APS) – Will cause irritation to eyes, which results in corneal injury, and to respiratory system.
May cause irritation to skin. Wear safety goggles, nitrile rubber gloves, gown and enclosed shoes.
-mercaptoethanol – Highly toxic. Toxic in contact with skin and if inhaled, harmful by inhalation and if swallowed.
Irritating to eyes, respiratory system and skin. Readily absorbed through skin. Combustible liquid and stench. Do not
breathe vapour. Wear chemical-resistant goggles, gloves (nitrile), gown and use only in a fumehood.
SDS- Respiratory, skin and eye irritant. Harmful if swallowed, inhaled or absorbed through the skin. Severe eye
irritant. Nuisance dust, but may also cause sensitization if inhaled. While working with SDS in solution, wear gloves,
gown, and mask where risk of splashing is possible. SDS is kept in small (<100ml) volumes in the lab.
Tris- Low toxicity- Skin, eye, and respiratory irritant. Decant in well ventilated areas. Wear gown and gloves when
handling.
Glycine- Low toxicity- eye irritant. Decant in well ventilated areas. Wear gown and gloves when handling.
Ponceau S – Irritant. Irritating to eyes, respiratory system and skin. Wear respirator, nitrile gloves, safety goggles, and
gown when handling.
Glycerol- Low toxicity. Glycerol may present a hazard with direct eye contact or prolonged skin contact. Wear gloves
and gown.
Coomassie Brilliant Blue- Low toxicity. Wear gloves and gown, and goggles if measuring volumes greater than 1g.
Power pac usage- All electrical items are hazardous. Because of the nature of this electrophoresis, there is a risk of
liquid (ie buffers) contacting the power pac. See “58_SWP_Working with electrical equipment_NC” and
“42_SWP_Powerpacks_RS”.
List emergency shutdown instructions
The 502 WW lab is equipped with a kill switch, located directly adjacent to the main entrance of the lab. This switch is within a box, as
entering the lab, on the right, 1.5m above ground level. If shutdown at the powerpoint of the BIO-RAD powerpacs is required but not
possible, the power to the entire lab can be shutdown by this kill switch.
There is an emergency exit leading to fire stairs from within the Rm501 lab.
The fire wardens: Justine Stehn, Anthony Kee
First Aid Officers in the lab: Christine Lucas, Renee Szokolai
List clean up and waste disposal requirements
Acrylamide gels are disposed of in cytotoxic waste containers. All buffers (Running Buffer, Transfer Buffer and Methanol) are
disposed of in designated labeled drums. No liquid waste from this procedure is disposed of in the sink, except ice water from the eski
surrounding the transfer tank. Used Whatman paper is disposed of as biological hazardous waste.
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List legislation, standards and codes of practice
used in the development of the SWP
NSW OHS Act 2000
NSW OHS Regulation 2001
Australia Dangerous Goods Code
Code of Practice for the Labelling of Workplace Substances
Code of practice for the Storage and Handling of Dangerous Goods
AS/NZS 2243.2:2006. Safety in laboratories. Part 2: Chemical aspects
Australian Standard AS2243.7-1991. Safety in laboratories. Part 7: Electrical Aspects.
AS/NZS 2161.1:2000 Occupational Protective Gloves – Selection, Use and Maintenance
UNSW Hazardous Waste Disposal Procedure
MSDS for all chemicals listed
Supervisory approval, training, and review
Supervisor: Peter Gunning
Signature:
Plant custodian:
Signature
List competency required – qualifications, certificates, licencing, training - eg course or instruction:
Training as per Training Needs Analysis, Induction into the Lab, Training on the SWP
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Safe Work Procedure
Date Effective: 01/01/2007
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Current Version: 1.2, 15/08/2007