OHS026
Safe Work Procedure
Faculty/Division
School/ Divisional Unit
Medicine
POWCS/ORC
Document number
Initial Issue date
POWCS.ORC.SWPM1 21/06/2006
Current version
3
Current Version
Issue date
Next review date
01/12/2010
29/02/2008
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:
SDS-PAGE and Western blotting (replaces M1.2)
Description:
Associated risk assessment title and location: POWCS.ORC.RA M1
Describe the activity or process
This SWP covers the protocol for performing Sodium Dodecyl Sulphate Polyacrylamide Gel
Electrophoresis (SDS-PAGE) and Western blotting. The use of numerous hazardous substances and
dangerous goods is involved. The attached Risk Assessment, all associated SWPs and all relevant
MSDSs must be read before this method is undertaken.
Procedure:
Combs and spacers are available in two thicknesses. For standard (thin) gels, prepare gels as described
below. When preparing thick gels, double the volumes described for the separating gel.
A. Gel preparation
1.
Assemble the glass plate sandwich as shown in Appendix 1. Take extreme care not to apply
excessive pressure when tightening the screws, as the glass plates will crack. Measure 5 cm from
the bottom of the plate and mark this level with a non-permanent pen. To confirm a good seal, fill
the glass plate sandwich with water and check for leaks. Tip off water before preparing the gel.
2.
In 2 x 50 mL tubes, prepare the separating (10% polyacrylamide) and stacking (4%
polyacrylamide) gels, using the following recipe. Add the reagents in this order, avoiding the
creation of bubbles. This is sufficient for 2 standard (thin) gels. It is a good idea always to prepare
at least two gels even if you only need one; you will have a backup if anything goes wrong, and
also you need to have a closed chamber (see Appendix 2). (NB: these are hazardous
substances)
40% acrylamide
Water
1M Tris-HCl pH 8.8
1M Tris-HCl pH 6.8
10% SDS
10% APS
Separating
2.5 mL
3.6 mL
3.75 mL
100 µL
50 µL
Stacking
1.0 mL
7.5 mL
1.25 mL
100 µL
50 µL
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3. Working in the fume cupboard, add 12.5 L TEMED to the separating gel only and mix once gently
by inversion. The acrylamide will begin to polymerise. Using a transfer pipette, fill the glass plate
sandwich to the level marked earlier. Overlay this with water-saturated butanol (the top layer is the
butanol). This helps the gel to set evenly. Do not discard any leftover separating gel yet, as it will serve
as an indicator as to when the gel has set (usually about 45 min). Any acrylamide waste should be
disposed of in the purple cytotoxic waste bins.
4. After the separating gel has set, remove the water-saturated butanol by tipping the whole assembly
over the sink, and then removing the last traces of butanol with a tissue. Rinse the top of the gel with
distilled water, which is discarded as described for butanol.
5. Next, working in the fume cupboard, add 12.5 L TEMED to the stacking gel and mix once gently by
inversion. The acrylamide will polymerise quickly so do not waste any time in loading the gel. Fill to the
top of the glass then insert the comb. Remove any bubbles that may have formed near the wells. Wipe
away any excess acrylamide solution with a tissue (dispose of correctly). Do not discard any leftover
separating gel yet as it will serve as an indicator as to when the gel has set (usually about 30 min).
B. Sample preparation
1. The standard (thin) 10-lane combs generally hold 20 L in volume; the thick 10-lane combs hold 50
L. Keep this in mind when preparing your samples, and also that you must add loading buffer.
2. Prepare samples in 0.5 mL tubes. Also prepare a tube with pre-stained protein standards (treat these
exactly the same as all test samples during preparation). Make all samples up to the same volume in
water to facilitate loading; for ease of calculation, it is simplest to dilute all samples to 15 L, in order
to allow for the addition of 5 L (when using a thin gel) of loading buffer. Loading buffer (which is
hazardous) consists of 4 
best to prepare this as a bulk mix and then add 5 µL to each sample tube. Heat the samples at 95 oC
for 5 min in a heating block. Cool on ice, then spin briefly to collect any condensation. The samples
are now ready to load provided the gel is set. If you are using a thick gel, you must scale up. Dilute
samples to 37.5 L in water, and add 12.5 L loading buffer (10 L sample buffer + 2.5 L DTT).
C. Gel Loading (NB: read SWP M3.3 before proceeding!)
1. Carefully remove the comb from the stacking gel. Rinse the wells with running buffer using a transfer
pipette. Remove the glass sandwich from the gel caster and assemble the upper buffer chamber as
shown in Appendix 2.
2. Place the unit into a gel tank and fill the wells with running buffer.
3. Load the samples into the wells, taking care not to carry any sample into neighbouring lanes. Gel
loading tips are very useful for this. (TIP: It is a good idea to practice your loading before running
actual samples! Just use water with 2 µL loading dye to visualise.)
D. Electrophoresis
1. After loading is complete, fill the inner chamber with running buffer first, then proceed to fill the
remainder of the tank until it is three-quarters full.
2. Place the lid (green) onto the tank and connect the electrodes (black to black and red to red). (NB:
this is an electric shock hazard)
3. Run at 100 V constant voltage until the dye front reaches the grey rubber seal. (Approximately 2 h;
alternatively, you may run at 150 V which takes just under 1 h.)
4. Keep an eye on the gel, as you will need to prepare items for the wet transfer procedure 10 min
before the gel finishes running.
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E. Wet-transfer (Blotting)
1. See Appendix 3 for items required in this section. You will need to have pre-cut nitrocellulose
(membrane) and pre-cut filter paper to the necessary size. The nitrocellose should be cut to 9 cm x 7
cm, while the filter paper should be slightly bigger (9.5 cm x 7.5 cm). (NB: nitrocellulose is
flammable)
2. Fill a plastic container with a couple of inches of 1X transfer buffer and prepare the transfer sandwich
in the buffer as follows on the black side of the cassette. (See Appendix 3.)
Fibre pad
Filter pad
Gel
Nitrocelloluse membrane (handle with forceps)
Filter paper (roll out any air bubbles that are present)
Fibre pad
(NB. You might find it easier to prepare the transfer sandwich on the clear side in the reverse order to
that described above:
Fibre pad
Filter paper (roll out any air bubbles that are present)
Nitrocelloluse membrane (handle with forceps)
Gel
Filter pad
Fibre pad
3. Close the cassette and place it in the electrode module, which is sitting in the gel tank (black to
black). Repeat steps 2 and 3 for other gel.
4. Obtain the cooling unit from the –20oC freezer in TC1 and place it in the tank.
5. Add the transfer buffer used in step 2 and if necessary add extra from the transfer buffer tank. Fill to
cover the top of the electrode module.
6. Transfer conditions: 250 mA (0.25 A) constant current for 45 min (NB: if preparing a thicker gel you
may increase the transfer time to 60-90 min to compensate). Upon completion, discard the filter
paper and, if the gel is no longer required, dispose of it in the purple cytotoxic waste bins. If the gel is
required, keep it hydrated or it will shrink. Store the gel at 4 oC in a square Petri dish containing PBST.
Place the blot (nitrocellulose) into a square Petri dish and then either proceed with detection of the
target protein or store in PBST at 4oC until ready.
F. Detection of target proteins
1. Rinse the blot once with PBS.
2. Prepare 5% skim milk powder (SMP) in PBST. Use 20 mL per blot. Block for 45 min at room
temperature with gentle rotation on the rotary platform shaker.
3. During this time, prepare your primary antibody. NB: incubation conditions will vary depending on the
supplier of the antibody. Generally for a primary antibody you can incubate in one of two ways; either:
i. Overnight at 4oC diluted in 20 mL 5% SMP in PBST with gentle rotation, or;
ii. 1 h at room temperature diluted in 20 mL 5% SMP in PBST with gentle rotation
4. Wash the blot 3 times with PBST (20 mL) with gentle rotation for 5 min, 10 min, and then 5 min.
During this time, prepare the secondary antibody (also in 5% SMP in PBST).
5. Incubate blots with the secondary antibody solution for 1 h at room temperature with gentle rotation.
6. Wash the blot 3 times with PBST (20 ml) with gentle rotation for 5 min, 10 min, and then 5 min.
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7. During this time, prepare the dark room for development of the film. Developing and fixative solutions,
and tap water for rinsing in between solutions, should be ready for use. The pre-labelled containers
that are used for these procedures are already in the dark room; there are two containers for tap
water. The developer and fixer are prepared as described above. These can be re-used several times
(until they grow dark).
8. Take the blot (still in PBST) to dark room and using forceps, place it on a paper towel to dry; the
protein standards should be facing up. Proceed to the signal amplification step.
G. Chemiluminescent signal amplification and detection
1. The kit is generally stored on the shelves at the back of the PC-2 laboratory (i.e. at room
temperature). Prepare the working solution in a square Petri dish. Generally you need about 2 mL per
blot; i.e. 1 mL Luminol/Enhancer solution plus 1 mL Stable Peroxide solution. Do not contaminate
either solution with its counterpart!! Use a new tip for each solution. Mix by tilting the dish.
2. Incubate the blot for 1 min in the working solution, then place it on a paper towel to dry, protein
standards facing up.
3. Wrap the blot in plastic wrap and place it into an x-ray cassette. Secure with tape to avoid movement.
4. Turn the red light ON and turn the white light OFF so as not to expose all the film in the box. Open
the box of autoradiography film and remove a single sheet, then re-seal the box and put it away.
Place the sheet of film over the blot and expose for the desired time. (NB: the time of development
will be determined by the strength of the signal, which in turn is determined by the amount of target
protein in your sample; the first time that you analyse a protein, it is usual to develop the film after ~ 1
min to check the strength of the signal; if necessary, a second - or third - film can be exposed, for
whatever length of time is necessary. However, the chemiluminescence system’s signal fades after 1
h, so this is the maximum period of exposure.)
5. Remove the film from the cassette and place in the container of developing solution. Agitate the
container gently until bands are visible.
6. Rinse in tap water.
7. Transfer the film to the container of fixative solution and again agitate. At this point, you may switch
the white light on. Don’t forget to turn the RED light off!
8. Transfer the film to the second container of tap water and rinse well, then hang up to dry.
9. Repeat steps 4 to 9 until the desired result is obtained.
10. Pour the used developer and fixer solutions back into their bottles when you are finished. Rinse out
all containers with water. (NB: do not pour old solutions down the sink! Label them with ChemAlert
labels and take them to the waste collection area at the rear of the main lab for removal by the waste
contractor.)
Supervisory approval, training, and review
Supervisor: Pamela J. Russell
Signature:
Plant custodian: Pamela J. Russell
Signature
List competency required – qualifications, certificates, licencing, training - eg course or instruction:
N/A
SWP review date: 22/01/09
Responsibility for SWP review: Elizabeth Kingsley
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List all resources required including plant, chemicals,
personal protective clothing and equipment, etc
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SDS-PAGE

40% Acrylamide/Bis solution ratio 37.5:1 (2.6%C) (BIORAD, Cat. # 161-0148; stored at 4C) (NB: this
is a hazardous substance)

Glass plates, one large, one small per gel (Astral Scientific, cat. # GBS07B-10S)

Combs and spacers (one and two, respectively, per gel)

Electrophoresis equipment

10% SDS solution (NB: SDS is flammable and hazardous, and at 10% is an irritant)

0.5 M Tris-HCl, pH 6.8 (NB: this is an irritant)

1.0 M Tris-HCl, pH 8.8 (NB: this is an irritant)

Water-saturated butanol (1:1 butanol:water) (NB: butanol is flammable)

TEMED (Sigma, cat. # T-9281) (NB: TEMED is flammable, toxic and corrosive)

10% Ammonium persulphate (APS; BIORAD, cat. # 161-0700) (prepare fresh in water or freeze at –
20oC) (NB: this is a hazardous substance)

Dithiothreitol (DTT; reducing agent; stored at -20C) (NB: this is a hazardous substance)

Protein markers (pre-stained, broad range; New England Biolabs, cat. # P7708S; stored at –20oC)

5X Sample buffer (200 mM Tris-HCl, pH 6.8, 8% SDS, 40% glycerol, 0.4% bromophenol blue) (NB:
this contains hazardous substances)

1X Running buffer (also called Laemmli buffer) (NB: this contains irritants)
Blotting

Wet transfer apparatus

Cooling unit

Nitrocellulose (0.45 µM, BIORAD, cat. # 162-0115)

Blotting paper

1X Transfer buffer
Protein Detection

1X PBS with 0.1% Tween-20 (PBST)

5% skim milk powder in PBST

Primary antibodies

Secondary antibodies (horseradish peroxidase-linked)

Rotary platform shaker
Chemiluminescent Signal Amplification and Development of Film

Supersignal® West Pico Chemiluminescent Substrate kit (Luminol/Enhancer solution and Stable
Peroxide solution) (Pierce, cat. # 34080) (NB: this contains an irritant)

Autoradiography film: Super RX medical X-ray film (Fujifilm)

Developing solution (Sigma cat. # P7042; Kodak GBX developer and replenisher; 103 mL to 370 mL
water) (NB: this is a hazardous substance)

Fixative solution (Sigma; cat. # P7167; Kodak GBX fixer and replenisher; 103 mL to 370 mL water)
(NB: this is a hazardous substance)
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List potential hazards and risk controls including specific
precautions required


SWP M3.3: Preparation of buffers for use in SDS-PAGE and Western Blot
MSDSs for:
- Acrylamide/Bis
- Sodium dodecyl sulphate (SDS)
- Tris
- Butanol
- TEMED
- Ammonium persulphate (APS)
- Dithiothreitol (DTT)
- Developing solution
- Fixing solution
- Chemiluminescence kit
List emergency shutdown instructions
List clean up and waste disposal requirements



SDS-PAGE gel material generated during the process have to be disposed of in the cytotoxic waste bin.
Transfer buffer containing methanol has to be collected in a large waste bin and disposed of as chemical
waste.
Kodak Developing and fixing solutions for the development of x-ray film has to be collected and disposed
of as chemical waste.
List legislation, standards and codes of practice used in
the development of the SWP
Not applicable
Supervisory approval, training, and review
Supervisor:
Signature:
Plant custodian:
Signature
List competency required – qualifications, certificates, licencing, training - eg course or instruction:
SWP review date:
Responsibility for SWP review:
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Appendix 1
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Appendix 2
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Appendix 3
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