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UNIVERSITY OF LEICESTER
CANCER STUDIES & MOLECULAR MEDICINE
Cell Signalling Section
STANDARD OPERATING PROCEDURE
SOP - 609
TITLE:
VERSION - 01
Western Blotting
Written by: Angie Gillies
Reviewed by: Lindsay Primrose
Changes introduced:
Implementation date: 02.05.08
Review date:
02.05.10
1. PURPOSE To provide a method for Western Blotting.
2. SPECIAL NOTES
Full details of the use of this procedure must be recorded in the appropriate laboratory
notebook, in accordance with SOP 105.
3. CROSS REFERENCES
3.1
3.2
3.3
3.4
3.5
3.6
Preparation of protein lysates
Preparation of conditioned media for Western blotting
Protein quantification
Preparation of gels for Western blotting
Polyacrylamide Gel Electrophoresis
Western Blotting
SOP 605
SOP 606
SOP 607
SOP 608
CPA 062
CPA 094
4. EQUIPMENT & MATERIALS
4.1
4.2
4.3
4.4
4.5
Gel apparatus and prepared gel
Molecular marker
Precision Plus Protein standards Dual Colour
Gel running buffer
Gel transfer buffer
Nitrocellulose membrane
4.6
4.7
4.8
4.9
4.10
4.11
3mm Filter paper
Gel loading buffer
PBS
TBS
Tween 20
Odyssey blocking buffer
4.12
Blocking agent (dried skimmed milk)
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Bio-Rad Mini-Protean 3
Bio-Rad 161-0374
RPM 112
RPM 113
Hybond-ECL
Amersham RPN2020D
Whatman3030700
RPM 115
RPM 7
RPM 30
Sigma P1379
LICOR Biosciences
927-40000
Marvel
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5. PROTOCOL
5.1
5.2
5.3
5.4
5.5
5.6
5.7
5.8
5.9
5.10
5.11
5.12
5.13
5.14
5.15
5.16
5.17
5.18
5.19
5.20
Remove the gel from the fridge. Unwrap and remove the comb.
Set up the gel apparatus. If only one gel to be run, use the buffer dam.
Transfer the gel apparatus to the electrophoresis tank. Fill both the top and
bottom with cold running buffer, ensuring the 2 buffers do not touch. Check
for any leaks.
Ensure there are no air bubbles at the base of the gel, as these would interfere
with the gel running. Using a syringe, carefully flush each well with buffer to
remove any debris and air bubbles.
Remove molecular marker from freezer and store on ice. It does not require
denaturing.
Prepared samples need to be denatured. Heat the samples at 99°C in a hot
block, for 5 minutes. Briefly centrifuge and store on ice.
Load the gel. Load 10µl of the molecular marker into well 1. If some of the
bands need to be run off, then load 20µl to ensure the remaining bands are not
too pale.
Load the samples. Load all of the sample, in 20µl aliquots. If the sample is
larger than 60µl it may be necessary to run the gel a little and then load the
remaining sample.
Connect the electrodes, black to black, and red to beige. (Negative to negative
and positive to positive.)
Run the gel with constant volts at 100-120V. The running time will be
approximately 1-4 hours, depending on the gel percentage and protein size.
The molecular marker colour bands can be seen as the gel runs.
If there is any leakage in the system, the top fluid layer will run to the bottom
layer. If this occurs, transfer some of the buffer from the bottom chamber to
the top chamber using a 10ml pipette. Ensure the electrodes are still touching
the buffer.
When the gel run is nearly complete, prepare the transfer materials. Cut a
piece of Hybond membrane to the gel size and place in a plastic tray
containing cold transfer buffer (containing methanol). Do not touch the
membrane with fingers, always use forceps.
Place the tray on an automatic shaker and leave for approximately 5 minutes.
Cut 2 pieces of 3mm filter paper the same size as the gel.
Set up the transfer tank. Pour cold transfer buffer into the tank just above the
minimum level.
When the gel has finished running, turn off the power and detach the
electrodes.
Carefully remove the gel(s) from the tank and drain off the solution.
Release the glass plates which are sandwiched together, by lifting the top plate
with the green wedge.
Carefully remove the gel and place into a plastic tray containing transfer
buffer. Wash for a few minutes to remove any residual SDS.
Place the gel on a clean glass plate. Cut away the upper phase of the gel
(wells) and discard.
Using forceps, place the saturated membrane on top of the gel. Cut to exact
size using a scalpel.
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5.21
5.22
5.23
5.24
5.25
Dampen one piece of filter paper with transfer buffer. Place onto the
membrane ensuring complete coverage.
Turn over the sandwiched membrane so the filter paper lies underneath.
Mark the membrane to identify the location of the markers. Use a pencil if
detecting with the LICOR system or a biro if using the ECL system.
Take the second piece of filter paper and dampen with buffer. Place directly
on the gel back. Use a pipette to gently roll out any air bubbles.
Dampen the sponges and place the gel/membrane sandwich into the transfer
cassette. The order should be:- Black side (Negative), sponge, filter
paper, gel, membrane, filter paper, sponge, beige side (Positive).
5.26
5.27
5.28
5.29
5.30
5.31
Close the plastic case and place in the tank with the clasp towards the top. The
electrodes are then black to black and beige to red.
Set up at 100 volts. If transferring during the day for approximately 1 hour,
room temperature is suitable. If transferring for more than 2 hours or
overnight, set up the system at 30 volts in the cold room.
Once transferred, carefully disassemble the cassette. Place the membrane in a
plastic tray containing either PBS (for LICOR detection) or TBS/Tween (for
ECL detection).
Note: the transfer buffer can be used twice. Collect and store at 4°C.
Blocking the membrane.
For LICOR detection:- use PBS/Odyssey blocking buffer at 1:1.
For ECL detection:- use 5% Marvel in TBS/0.1% Tween 20.
Both must be prepared fresh.
Drain off the PBS or TBS/Tween and add the appropriate blocking solution.
Blocking requires at least 1 hour at room temperature or overnight at 4°C, on a
shaker. 300ml of blocking solution is needed to block 2 membranes.
Proceed to SOP 610 for detection of the proteins.
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