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Western Immunoblotting

Preparation of protein

Cell lysed to release proteins and the protein concentration calculated using
BCA. Volume and type of lysis buffer can differ depending on cell pellet size
and the nature of the protein of interest.

1. Fast cool centrifuge @ 4
O
C
2. Resuspend cell pellet in 500-1000l of lysis buffer
3. Leave on ice for 5 mins
4. Spin 16000g (rcf) for 40mins @4
o
C

5. Make standards for BCA (Pierce BCA Protein Assay Kit Thermo).
Appendix 1
6. Pipette 25l of each standard into well of 96-well plate (pipette up in
concentration I to A so do not have to change pipette tip)
7. Samples removed from centrifuge and transferred into new Eppendorf
tube without disturbing the cytoskeleton at the bottom of the tube
8. Pipette 25 l of sample into the 96-well plate
9. To start the BCA, reagents A and B made into a master mix (samples +
standards + extra 200l per well) at 1:50 B:A.
10. Incubate 96-well plate for 30mins @37
o
C
11. Read absorbance at 562nm









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Preparation of gel

Two gels are made, the stacking gel and the resolving gel. Percentage of the
resolving gel differs due to the size of protein. A higher percentage is needed
for smaller proteins.
1. Clean short and spacer plates and set up as appendix 2
2. Make resolving and stacking gel in 15ml falcon tube


3. APS and TEMED added to resolving gel and then pipetted between the two
plates
4. Pipette ~1ml isopropanol onto resolving gel to level gel out
5. Once gel has set remove isopropanol using filter paper
6. Add water to clean and remove using filter paper
7. Add APS and TEMED to stacking gel
8. Pipette stacking gel on top of the resolving gel and insert comb
9. Once gel has set put in tank (appendix 3) and fill with 1x running buffer
10. Stored o/n @4
o
C


Resolving

1. Master mix of DTT and loading buffer :
a. Loading buffer (3x) - 10l per sample (for loading 30l total)
b. DDT (20x) 1.5l per sample (for loading 30l total)
2. Add amount of sample needed (calculated from BCA) and the appropriate
lysis buffer to Eppendorf tube
3. 11.5l of the DDT and loading buffer master mix to each of the protein
samples
RESOLVING GEL (12%) STACKING GEL
3.45ml dH2O 2.9ml dH20
2.4ml 40% Acrylamide 0.75ml 40% Acrylamide
2ml 1.5M Tris (pH 8.8) 1.25ml 0.5M Tris (pH 6.8)
80l 10% SDS 50l 10% SDS
Add APS and TEMED last
80l 10% APS 50l 10% APS
8l TEMED 5l TEMED
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4. Denature protein on heat block for 10mins @90
o
C
5. Quick spin down
6. Load 10l of protein ladder
7. Load 30l of each sample
8. Run at 90v whilst samples move through stacking gel
9. Once through stacking gel, STOP and run at 120v until run through
resolving gel
Transferring

1. Black side of cassette down. Sponge in cassette first followed by filter
paper cut to size (2x 10cm by 7cm)
2. Remove gel from glass plates and cut off stacking gel and blue bands at
the bottom of the gel
3. Place gel onto the filter paper in cassette
4. Nitrocellulose membrane over the gel and cut to size of gel
5. 2
nd
filter paper over the membrane
6. Sponge on last before clamping the cassette together
7. Insert cassette into tank (black to black)
8. Fill tank with 1x transfer buffer (10/20% MeOH) and add ice block
9. Run at 250mA for 2 hours

Membrane blocking and Antibody Incubation

1. Ponceau stain over membrane to visualises total protein. Reusable
2. Wash off in water
3. Transfer membrane into a 50ml falcon tube with ~10ml of 5% blocking
buffer (BSA or milk) (1.25g in 25mls TBST)
4. Incubate membrane in blocking buffer for one hour at room temperature
on rocker
5. Remove blocking buffer, leaving in required amount for antibody dilution
6. Add primary antibody to the blocking buffer and incubate O/N @4
o
C on
rocker

7. TBST washes, 1x 15 mins, 1 x 10 mins, 1 x 5 mins
8. Add required amount of milk and HRP labelled secondary antibody
9. Incubate at room temperature on rocker for 1 hour
10. TBST washes, 1x 15 mins, 1 x 10 mins, 1 x 5 mins
11. ECL prime western blotting detection reagents mixed in a 15ml falcon
tube wrapped in foil. 1:1 1ml of both reagent A and B
12. Pipette ECL mix onto blot and agitate for 5 mins
13. Transfer blot to plastic sheet for imaging
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Loading control

1. TBST washes, and 1x 15 mins, 1 x 10 mins, 1 x 5 mins
2. Add loading control antibody in milk for 1hr at room temperature on
rocker (anti - tubulin 1:1000)
3. TBST washes, 1x 15 mins, 1 x 10 mins, 1 x 5 mins
4. Add secondary antibody and incubate at room temperature for 1hr on
rocker
5. TBST washes, 1x 15 mins, 1 x 10 mins, 1 x 5 mins
6. ECL prime western blotting detection reagents mixed in a 15ml falcon
tube wrapped in foil. 1:1 1ml of both reagent A and B
7. Pipette ECL mix onto blot and agitate for 5 mins
8. Transfer blot to plastic sheet for imaging













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Appendix 1 BCA Standards

STANDARD BSA (L) LYSIS BUFFER (L) CONCENTRATION
(G/ml)
A 300 BSA - 2000
B 375 BSA 125 1500
C 325 BSA 325 1000
D 175 VIAL B 175 750
E 325 VIAL C 325 500
F 325 VIAL E 325 250
G 325 VIAL F 325 125
H 100 VIAL G 400 25
I - 400 BLANK













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Appendix 2 Biorad mini protean 3 cell set up




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Appendix 3 Biorad mini protean 3 cell set up

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