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Author: Michelle Farquhar/Garrick Wilson
SDS-PAGE and Western Blotting
Solutions:

Acrylamide/Bis (30% T, 2.6% C) Sigma A3699
Available from stores
Acrylamide/Bis-acrylamide
30%solutions 37.5:1 IBR-A3699 £28.95

10 % (w/v) SDS (Sigma L3771-100G)
Dissolve 10 g SDS in 90 ml dH
2
O with gentle stirring and bring to 100 ml with dH
2
O.

Resolving Gel buffer 1.5 M Tris-HCl, pH 8.8
27.23 g
100 ml
Tris base dH
2
O
Dissolve Tris base in 80 ml dH
2
O, adjust to pH 8.8 with HCl.
Bring total volume to 150 ml with dH
2
O.
Store @ 4 o C – warm to room temperature prior to use.

Stacking Gel buffer 0. 5M Tris-HCl, pH 6.8
6 g
100 ml
Tris base dH
2
O
Dissolve Tris base in 60 ml dH
2
O, adjust to pH 6.8 with HCl.
Bring total volume to 100 ml with dH
2
O.
Store @ 4 o C – warm to room temperature prior to use.

Sample buffer (SDS reducing buffer)
3.55 ml
1.25 ml
2.5 ml
2.0 ml
0.2 ml dH
2
O
0.5 M Tris-HCl, pH 6.8 glycerol
10 % (w/v) SDS
0.5% (w/v) bromophenol blue (Sigma B5525-5G)

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9.5 ml Total volume
Store @ room temperature.
Use: Add 50 ul

-Mercaptoethanol to 950 ml sample buffer prior to use.
Dilute the sample at least 1:2 with sample buffer and heat at 95 o C for 4 min.
(n.b. for non-reduced samples DO NOT add

-Mercaptoethanol substitute with 50 ul dH
2
O)

10x Electrode (Running) Buffer pH8.3 (Makes 1 L)
30.3g
144.0g
Tris base
Glycine (Sigma G8898-500G)
10.0g SDS
Dissolve and bring total volume to 1000ml with dH
2
O
DO NOT adjust pH with acid or base.
Store @ 4 o C
– warm to room temperature prior to use.
Use: dilute 50ml of 10x stock with 450ml dH
2
O

10 % APS (fresh daily) (Sigma A3678-25G)
100 mg ammonium persulphate
Dissolved in 1 ml of dH
2
O

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Gel formulations 10 ml:
Prepare the monomer solution by mixing all reagents except TEMED and 10
% APS.
Degas the mixture for 15 min (optional).
Percent gel
%
4
5
DDI H
(ml)
6.1
5.7
2
O 30 % Degassed
Acrylamide/Bis (ml)
1.3
1.7
*Gel buffer
(ml)
2.5
2.5
10 % w/v
SDS (ml)
0.1
0.1
6
7
8
9
10
11
12
13
14
5.4
5.1
4.7
4.4
4.1
3.7
3.4
3.1
2.7
2.0
2.3
2.7
3.0
3.3
3.7
4.0
4.3
4.7
2.5
2.5
2.5
2.5
2.5
2.5
2.5
2.5
2.5
15
16
2.4
2.1
5.0
5.3
2.5
2.5
0.1
0.1
17 1.7
* Resolving Gel Buffer
* Stacking Gel Buffer
Immediately prior to pouring gel add:
5.7 2.5 0.1
For 10 ml monomer solution
Resolving gel
5 ul
50 ul 10 % APS
TEMED (Sigma T9821-25ml)
Stacking Gel 50 ul
10 ul
10 % APS
TEMED
Swirl gently to initiate polymerisation
Note: Prepare any desired volume of monomer solution by using multiples of
10ml adjusting the APS and TEMED volume accordingly.
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1

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
Transfer buffer (1 L)
25 mM Tris pH 8.3, 192 mM glycine, 20 % v/v MeOH, 0.05 % SDS
3.03 g
14.4 g
Tris base
Glycine
200 ml
500 ul
MeOH
10 % SDS
Add dH
2
O to 1000 ml.
DO NOT adjust pH with acid or base.
Store @ 4 o C.

TST, washing/Ab/blocking buffer (1 L) pH 7.5
I 10 mM Tris, 100 mM NaCl, 0.05 % w/v Tween 20
1.21 g Tris base
5.84 g
0.5 ml
NaCl
Tween 20
Dissolve in 900 ml dH
2
O, adjust to pH 7.5 with HCl.
Bring total volume to 1000 ml with dH
2
O.
Store @ 4 o C
– warm to room temperature prior to use.
For blocking and Ab incubations add Marvel to 5 % w/v
5 g / 100 ml TST

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SDS-PAGE
Mini-PROTEAN 3 Cell BIO-RAD: 165-3301
Glass cassette and casting stand assembly:
1. Ensure plates are clean and dry prior to use.
2. Place casting frame upright with pressure cams in the open position facing forward on a flat surface.
3. Select spacer plate of desired gel thickness (0.75 mm) and place short plate on top of it.
4. Orient the spacer plate so the labelling is up and facing forward.
5. Slide the two glass plates into the casting frame, keeping the short plate at the front.
6. When the glass plates are in place, engage the pressure cams to secure the glass cassette sandwich in the casting frame. Note: ensure plates are flush on a level surface.
7. Engage the spring loaded lever and place the gel cassette assembly on the grey casting stand gasket. The lever pushes the spacer plate down against the grey rubber gasket.
Gel casting:
1. Place a comb completely into the assembled gel cassette. Mark the glass plate 1cm below the comb teeth. This is the level to which the resolving gel is poured. Remove the comb.
2. Prepare the resolving gel monomer solution by combining all reagents except APS and TEMED. Degas solution.
3. Add APS and TEMED and pour to the mark using a disposable plastic pipette.
4. Immediately overlay the solution with isopropanol.
5. Allow gel to polymerise for ~45 min.
6. Rinse the gel surface with dH
2
O.
7. Prepare the stacking gel monomer solution by combining all reagents except APS and TEMED. Degas solution.
8. Add APS and TEMED and pour to the top of the short plate using a disposable plastic pipette.
9. Insert the desire comb between the spacers.
10. Allow to polymerise for ~30 min.
11. Gently remove comb and rinse wells with running buffer.
12. Gel can be stored at room temperature overnight in running buffer.
13. Rinse the casting frame and stand with dH
2
O. Wash glass plates with a mild 1% Trigene solution and rinse well, spray with 70 % EtOH and allow to dry.
Module assembly and sample loading:

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1. Remove gel cassette sandwich from casting stand by rotating cams inwards to release the plates.
2. Place gel cassette sandwich into slots at bottom of each side of the electrode assembly. Ensure the short plate faces inwards to fit under the green gaskets.
3. Slide electrode assembly into clamping frame and close to seal and form inner chamber.
4. Lower inner chamber into the mini-tank (outer chamber).
5. Fill inner chamber with ~125 ml running buffer until level reaches half way between tops of spacer and short plates.
6. Add ~200 ml running buffer to the mini-tank (outer chamber).
7. Load samples into wells using gel loading tips. Each well will accept 25 ul maximum volume.
8. Load 10 ul Rainbow Molecular Weight Marker (Amersham RPN800).
9. Load unused wells with 10 ul sample buffer to ensure lanes run symmetrically.
Gel Electrophoresis:
1. Place lid on mini-tank. Align colour coded plugs and jacks.
2. Insert electrical leads into power pack checking colour coded polarity.
3. Run the gel at 200 volts constant for approximately 35 min .
4. Check where dye front has run to ~1 cm above bottom of gel (if necessary can be run for longer to reach this point).
5. Turn of power supply.
6. Remove tank lid pour off buffers.
7. Open the inner chamber and remove electrode assembly.
8. Open electrode assembly and remove gel cassette sandwich.
9. Remove gels from gel cassette sandwich by gently separating the two plates using the plastic gel releaser.
10. Run the gel releaser along each edge of the spacer plate to separate the gel from the plate.
11. Remove the gel by floating it off the glass plate by inverting the gel and plate under transfer buffer.
12. Rinse all equipment after use in dH
2
O.

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Western Blotting

Fill the Bio-Ice cooling unit with water and store in freezer at -20 o C until ready to use. After use re-fill and return to freezer.

Prepare transfer buffer (using buffer chilled to 4 O C will aid heat dissipation).

Cut the PVDF membrane (Sigma P2938-ROL) and filter paper to the required dimensions.
PVDF 6.5 x 8.5 cm
Filter paper7.5 x 10.5 cm

Soak PVDF for ~1 min in MeOH and rinse with water. Always wear gloves when handling membrane.

Equilibrate gel and soak the membrane, filter paper and fibre pads in transfer buffer for ~15 min.

Prepare the gel sandwich:
1. Place cassette with black side down on a clean surface.
2. Place one pre-wetted fibre pad on grey side of cassette.
3. Place sheet of filter paper on the fibre pad.
4. Place the equilibrated gel on the filter paper (removing any air bubbles).
5. Place the pre-wetted PVDF membrane on the gel (removing any air bubbles).
6. Place a piece of filter paper on the membrane.
7. Place one pre-wetted fibre pad on the filter paper.
Clear side of cassette
Fibre pad
Filter paper
PVDF membrane
Gel
Filter paper
Fibre pad
Black side of cassette
TIPS :
BENCH
To avoid mirror image place gel face down so that transfer onto membrane is in order of how loaded originally
Snip upper right corner of membrane and again place face down on top of gel to orientate membrane for detection

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
Close the cassette and lock with white latch. Place cassette in transfer module with black side of cassette against black side of transfer module.

Place in tank with magnetic stirrer bar and Bio-Ice cooling unit.

Fill tank with transfer buffer and place on magnetic stirrer at maximum speed.

Place lid on tank using colour coding.

Run transfer at 350 amps constant for 60 min .

Upon completion turn of power supply, pour off buffer disassemble blotting sandwich and remove membrane.

Rinse all equipment in dH
2
O.
Antibody detection:

Ensure rainbow marker has transferred to membrane.

Place membrane in blocking solution (5 % Marvel/TST) and incubate for
1 h with agitation (this can be left at 4 o C overnight or over a weekend if necessary).

Discard solution.

Add primary antibody in blocking solution and incubate for 1 h with agitation (2ml total volume in a falcon tube).

Wash membrane in 50 ml TST 5x 5 min.

Add secondary antibody in blocking solution and incubate for 1 h with agitation (2ml total volume in a falcon tube).

Wash membrane in 50 ml TST 5x 5 min.

Prepare ECL by combining equal volumes of ECL solutions A and B
(Geneflow KI-0170). Note: use different tips in each bottle as cross contamination will render solutions useless.

Place membrane in ECL for ~30 sec.

Remove and wrap in cling film protein side uppermost.

Place in developing cassette.

Expose film to membrane for desired period of time and develop.
Tip: 30 sec exposure should be optimal

Store membrane wet in cling film at 4 o C.
Note:
Different antibodies will require different incubation times more so with the primary antibody which may require overnight.
Some antibodies prefer a different blocking solution such as 3% BSA overnight.

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Stripping and reprobing membrane:
Stripping buffer
62.5 mM Tris-HCl (pH 6.7)
100 mM

-Mercaptoethanol
2 % (w/v) SDS

Place membrane in a falcon tube and add 50 ml stripping buffer.

Incubate at 50 o C for 30 min with occasional agitation.

Wash membrane 4x 5 min in TST.

Block membrane and perform immunodetection as standard protocol.