2008

PROTOCOLS FOR GEL ELECTROPHORESIS AND WESTERN

BrainNet Europe II
BLOTTING
Project Co-ordinator:
These protocols are used for current practice in many laboratories. The Prof. Dr. Hans Kretzschmar
standard protocol is followed by minor modifications used in other
laboratories. All of them have been tested with good results, and preferences
are largely dependent on personal feelings. Contact:
www.brainnet-europe.org

SAMPLE HOMOGENIZATION
Approx. 0.1 grs of fresh tissue in 4 volumes of buffer:
* Lysis buffer: 20 mM HEPES-KOH pH = 7.5
250 mM sucrose Network of Excellence funded
10 mM KCl by the EC in the 6th Framework
Program “Life Science”
1.5 mM MgCl2
1 mM EDTA
1 mM EGTA
1mM DTT
0.1 mM PMSF
a table of proteases inhibitors (Roche) in 10 mL of lysis
buffer Content
* Centrifuge 15,000xg 5 min. PROTOCOLS FOR GEL
ELECTROPHORESIS AND WESTERN
BLOTTING ...........................................1
* Remove the supernatant carefully SAMPLE HOMOGENIZATION ............1
PROTEIN QUANTIFICATION:
BRADFORD (1l)...................................1
SAMPLE BUFFER (2X) .......................2
PROTEIN QUANTIFICATION: BRADFORD (1l) ELECTROPHORESIS .........................2
RUNNING BUFFER (10x)....................3
0.1 grs Comassie Brilliant SEMI-DRY TRANSFER .......................3
SEMI-DRY BUFFER (500ml)...............3
12.5 ml ethanol 95º SANDWICH TRANSFER .....................3
IMMUNOBLOTTING ............................6
MEMBRANE STRIPPING....................7
100 ml phosphoric acid at 85% MODIFICATIONS TO THE
STANDARD PROTOCOL ....................7
850 ml H2O miliQ PROTOCOL EDINBURGH ..................7
WESTERN BLOT:
PROTOCOL MILANO ..........................9
TWO-DIMENSIONAL GEL
Once the protein is quantified from the supernatant, dilute in sample buffer at ELECTROPHORESIS .......................14
a ratio of 1:1 DETAILED 2D PROTOCOL...............14
MASS SPECTROMETRY COMPATIBLE
SILVER STAIN PROTOCOL .............18

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SAMPLE BUFFER (2X)
1.514 grs Tris Base in 25 ml miliQ H2O (125mM), adjusted pH=6.8 with HCl
40 ml SDS 10%
20 ml glycerol
0.002 grs Bromophenol Blue
Add miliQ H2O until 100 ml
β-mercaptoethanol at 10% is added to the samples just before used.

Heat the sample at 95% for 5 min. Keep at -20ºC until use.

ELECTROPHORESIS
RESOLVING SOLUTION

STACKING SOLUTION

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Buffer R: 1.5M Tris HCl PH= 8.8
0.1% SDS
Buffer S: 0.5M TrisHCl PH= 6.8
AP: Ammonium Persulfate: 0.1 mgrs/ml H2O miliQ

RUNNING BUFFER (10x)

30.28 grs/l Tris (0.25M)
14.13 grs/l glycine (1.92M)
10 grs/l SDS (0.1%)
Conditions: constant current, 0.02A per gel

SEMI-DRY TRANSFER
4 whatman papers + nitrocellulose membrane + gel + 4 whatman papers
(all embedded in semi-dry buffer)
Conditions: constant current, 0.04A per gel, 45 minutes.

SEMI-DRY BUFFER (500ml)

2.9 grs Tris
1.45 grs glycine
100 ml methanol
0.19 grs SDS

Alternatively (depending on the molecular weight of the protein):

SANDWICH TRANSFER
Sandwich buffer
250 mM Tris base
92 mM glycine
in 1 l of distilled water.

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Add 10% of methanol (if proteins <30 kDa) or 20 % (if proteins >30 kDa) just
before use.
(This protocol has been prepared for using the Mini-Trans blot Electrophoretic
Transfer
cell (Bio-Rad))
• Fill the Bio-Ice cooling unit with water and store it in your laboratory
freezer at -20 °C until ready to use. After use, return the cooling unit to
the freezer for storage.
• All electrophoresis gels should be pre-equilibrated in transfer buffer prior
to electrophoretic transfer. Pre-equilibration will facilitate the removal of
contaminating electrophoresis buffer salts and neutralization salts (salts
resulting from the denaturation of nucleic acids prior to transfer). If the
salts are not removed, they will increase both the conductivity of the
transfer buffer and the amount of heat generated during the transfer.

Protocol:
1. Prepare the transfer buffer. (Using buffer chilled to 4 °C will improve heat
dissipation.)
2. Cut the membrane (a) and the filter paper to the dimensions of the gel.
Always wear gloves when handling membranes to prevent contamination.
Equilibrate the gel and soak the membrane, filter paper, and fibre pads in
transfer buffer (15 min–1 hour, depending on gel thickness).
3. Prepare the gel sandwich. Place the cassette, with the grey side down, on a
clean surface.
Place one pre-moistened fibre pad on the grey side of the cassette.
Place a sheet of filter paper on the fibre pad.
Place the equilibrated gel on the filter paper.*
Place the pre-moistened membrane on the gel.*
Complete the sandwich by placing a piece of filter paper on the membrane.*
Add the final fibre pad.
* Removing any air bubbles which may have formed is critical for good
results. Use a
glass tube to gently roll air bubbles out.
(a) Nitrocellulose or PVDF membranes can be used. PVDF (Polyvinylidene
difluoride) membranes must first be moistened in 100% MetOH.

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4. Close the cassette firmly, being careful not to move the gel and filter paper
sandwich.
Lock the cassette with the white latch.
5. Place the cassette in the module. Repeat for the other cassette.
6. Add the frozen Bio-Ice cooling unit. Place in tank and completely fill the
tank with buffer.
7. Add a standard stir bar to help maintain even buffer temperature and ion
distribution in the tank. Set the speed as fast as possible to keep ion
distribution even.
8. Put on the lid, plug the cables into the power supply, and run the blot.
Transfer conditions: 200 mA per gel for 90 min. Voltage must not exceed
120V.
9. Upon completion of the run, disassemble the blotting sandwich and remove
the membrane for development. Clean the cell, fibre pads, and cassettes with
laboratory detergent and rinse well with de-ionized water.
*When the membrane is transferred, check the transference efficiency with
Ponceau staining. Mark the molecular weight standards and rinse the
membrane with distilled water to take out the dyer.

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IMMUNOBLOTTING
Reagents

1. TBST 10X (Tris-Buffered Saline) buffer

Trizma base 12.11 g
NaCl 81.81g
dH2O 1000 mL
Adjust pH to 7.4 with HCl
Add 1% Tween-20.

2. Blocking solution
5% skimmed milk in TBST

3. Primary antibody
Dilute the primary antibody with TBST-BSA3%

4. Secondary antibody (prepare before use)

Dilute the secondary antibody 1:1000 with blocking solution

Protocol
1. Block the membrane for 1h with blocking solution at room temperature.
2. Wash the membrane with TBST 3 times, 5 minutes each.
3. Incubate the membrane with the primary antibody at the appropriate
dilution overnight at 4ºC.
4. Repeat step 2.
5. Incubate the membrane with the secondary antibody for 45 minutes at room
temperature.
6. Wash the membrane with TBST for 30 minutes as a minimum, changing the
buffer each 5 or 10 minutes. Longer washing times will increase the efficiency
of the detection.

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7. Different detection methods can be used, depending on the sensitivity and
the specificity of the antibodies used. In general a chemiluminescent method is
used to detect the reactive bands following the instructions given by the
manufacturer.

MEMBRANE STRIPPING
To re-utilize the membranes with another primary antibody
Stripping buffer
3.78 grs of Tris in 500 ml distilled H2O. Adjust pH=6.8 with HCl
10 grs SDS (2%)
3.48 ml β-mercaptoethanol
Wash membranes with TBST
Incubate the membranes twice for 20 min. each time with stripping buffer at
66ºC, approx.
3 washes for 5 min. each of TBST
Repeat the immunoblot from blocking of the non-specific unions.

MODIFICATIONS TO THE STANDARD PROTOCOL

PROTOCOL EDINBURGH
Protein Extraction and Quantification
• Approx. 0.1g frozen brain tissue was placed into sterile eppendorfs
containing 1mL lysis buffer:
• Lysis Buffer:
o 20 mM HEPES-KOH, pH 7.5
o 250 mM sucrose
o 10 mM KCl
o 1.5 mM MgCl2
o 1 mM EDTA
o 1 mM EGTA
o 1 mM DTT

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 o 0.1 mM PMSF
• Sigma Protease Inhibitor Cocktail (10 l) was added to each sample.
• Samples were lysed at 4oC for 1 hr and were vortexed intermittently.
• Centrifuge at 13,000 rpm for 20 min and remove supernatant to a new
sterile eppendorf.
• Samples were diluted 1:1 in sample buffer and heated at 95oC for 5 min
prior to use.
• Sample Buffer:
o 1.25 ml 0.5M Tris-HCl, pH6.8
o 2.5 ml glycerol
o 2 ml 10% SDS
o 0.2 ml 0.5% Bromophenol Blue
o 3.55 ml dH2O
Add 50 μL mercapto to 950 μL buffer prior to use.

Electrophoresis
• 10% resolving gel and 4% stacking gel on Mini Protean 3 system from
Biorad. However, was run at 100V, constant current
• High Molecular Weight Rainbow Markers (Amersham)
• Approx. 50 g protein per well.

Sandwich Transfer
• Was run using Mini TransBlot Electrophoretic Transfer Cell (Biorad)
• 2 filter pads + gel + PVDF (Immobilon) + 2 filter pads
• Sandwich buffer with 20% methanol
• Transferred at 100V for 1 hr

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Immunostaining
• Membrane was equilibrated in TBST for 15 min then blocked in TBST-
4% BSA for 1 hr on a rocker, RT
• Primary antibody (b-actin 1:5000; phosphor-AKT 1:1000) was added and
incubated at 4oC overnight on a rocker.
• 3 x 10 min washes in TBST-4% BSA
• Biotinylated secondary antibodies were added at 1:2000 (b-actin – rabbit
anti mouse, DAKO; phosphor-AKT- swine anti-rabbit, DAKO) and
samples incubated at RT for 1 hr.
• 1 x 10 min wash in TBST-4% BSA, 2 x 10 min washes in TBST
• Streptavidin-ABC (DAKO) diluted 1:50 was added and samples
incubated for 30 min at RT
• 3 x 10 min washes in TBST
• Develop with Vector VIP peroxidase substrate kit for 15 min. Wash in
dH2O for 5 min. Dry membranes.

WESTERN BLOT: PROTOCOL MILANO

Lysis buffer normally used:
NaCl 100mM
EDTA 10mM
NP40 0.5%
NaDeoxic 0.5%
Tris pH7.4 10mM
PEFABLOC 1mg/ml
EDTA 0.5mg/ml
LEUPEPTINE 10μg/ml
PEPSTATINE 10μg/ml
APROTININE 1 μg/ml

Centrifugation 15,000xg 5 min.

Remove the supernatant carefully

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PROTEIN QUANTIFICATION: BCA PROTEIN ASSAY (PIERCE)
0.1 grs Comassie Brilliant
12.5 ml ethanol 95º
100 ml phosphoric acid at 85%
850 ml H2O miliQ

Once the protein is quantified, dilute in sample buffer at a ratio of 1:1

SAMPLE BUFFER (2X)

SDS 4% → 40ml
TRIS pH6.8 120mM → 1.454g
BB 0.002% → 0.002g
GLYCEROL 20% → 20ml
DW until 100ml
DTT 100mM

Heat the sample at 95% for 10 min. Keep at -20ºC until use.

ELECTROPHORESIS
RESOLVING

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Buffer R: 1.5M Tris HCl PH= 8.8
0.1% SDS
Buffer S: 0.5M TrisHCl PH= 6.8
Ammonium Persulfate: 0.1 mgrs/ml H2O miliQ

RUNNING BUFFER (10x)

30.28 grs/l Tris (0.25M)
14.13 grs/l glycine (1.92M)
10 grs/l SDS 1%
Conditions: constant voltage

SEMI-DRY TRANSFER
4 whatman papers + PVDF membrane + gel + 4 whatman papers
(all embedded in semi-dry buffer)
conditions: constant current

SEMI-DRY BUFFER (500ml)

2.9 grs Tris
1.45 grs glycine
100 ml methanol
0.19 grs SDS

Alternatively (depending on the molecular weight of the protein):

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SANDWICH TRANSFER
2 whatman papers + gel + nitrocellulose membrane + 2 whatman papers
(all embedded in sandwich buffer)

Transfer conditions

TBST BUFFER (10x)

12.11 grs (100 mM) Tris
81.81 grs (1.4 M) NaCl

Adjust pH= 7.4 with HCl and then add 1% Tween-20

Add miliQ H2O to 1 l

Blocking of non-specific unions: 1 h with TBST+ 5% skimmed milk

Primary antibody inTBST-milk. Overnight at 4ºC
3 washes for 10 min in TBST
Secondary antibody diluted in TBST-milk at 1:3000 (DAKO) for 60 min
3 washes for 10 min in TBST
3 washes for 5 min. each in TBST
Develop with ECL (Amersham)

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PHF PURIFICATION PROTOCOL
Protocol used to obtain cellular fractions enriched in paired helical filaments
and related filaments in Alzheimer’s disease and tauopathies
Tissue homogenisation (approx 2-3 grs of tissue) 1/10 (w/v)

Buffer H: 10mM Tris-HCl (PH= 7.4)

0.8M NaCl
1mM EGTA
10% sucrose
1mM PMSF
protease inhibitor cocktail

Determination of the protein concentration with BCA method (Pierce)

Electrophoresis (load approx.100 μgrs of protein) and Western blot.

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TWO-DIMENSIONAL GEL ELECTROPHORESIS
Brain tissue (0.1g) suspended in 1 mL of extraction buffer consisting of 40
mM Tris-HCl, 7M urea, 2M thiourea, 4% CHAPS and a mixture of protease
inhibitors (Sigma). The suspension is sonicated for approximately 30 sec
(except if the sample is prepared for oxydized proteins) and centrifuged at
12,000 rpm for 15 min. Sonication can produce a kind of oxidative stress,
exaggerating the final results. The protein content in the supernatant is
determined with the Bradford method. 300 μg of protein sample are diluted in
extraction buffer plus 2 mM TBP (Tributylphosphine) and 0.2% carrier
ampholytes pH 3-10 applied (300 μL) on immobilized pH 3-10 linear gradient
strips (17 cM). The re-hydration step is done overnight passively. The proteins
are focused at 300 V for 1h, after which the voltage is gradually increased to
3,500 V within 6 h. Focusing is continued at 3,500 V for 12 h and at 5,000 V
for 24 h. Before the second dimension starts, the strips must be equilibrated
first in equilibration buffer plus 2% DTT for 20 minutes, and afterwards in the
same buffer but now containing 2.5% iodoacetamide, for 20 min. Finally, the
strip is soaked in SDS-PAGE running buffer for 30 sec and then placed onto
the gel. The second-dimensional separation is usually performed on 10%
polyacrylamide gels at 200 V for 4-5 h. After protein fixation with 40%
methanol containing 10% acetic acid in de-ionized water for 1h as a minimum,
the gels are stained with the Silver Staining kit (Amersham-Pharmacia),
following the protocol supplied by the manufacturer.

DETAILED 2D PROTOCOL
1. PROTEIN PRECIPITATION
Buffer interference with the first dimension must be avoided. A way to
achieve this is by protein precipitation followed by solution in 2D-sample
buffer.
Precipitation buffer (PB)
Tris-Bufferd Saline (TBS) buffer 10mM pH 7.4.
Ethylenediaminetetraacetic (EDTA) 1mM
Sodium Pyrophosphate 5mM
β-glycerolphosphate 30 mM
Sodium Fluoride 30 mM

(May be prepared and kept at –20ºC)

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(The starting amount is not important. What is important is the ratio of sample
to PB).
1. Homogenize 0.1g of brain sample in 4-5 volumes of PB.
The amount of protein is quantified by the BCA method using BSA as a
standard. The same amount of protein from each sample is used to precipitate.
2. Four volumes of HCl 2N are added to each sample and incubated for 20
minutes at room temperature.
3. Tri-chloroacetic acid (TCA) at 15% final volume is added to each sample
and incubated on ice for 10 minutes.
4. Vortex the samples to mix well.
5. Centrifuge 15,800 g, 2 minutes. Supernatants are to be discarded.
6. Wash the pellets 3 times in ethanol-ethylacetate (1:1). These final pellets are
the precipitated proteins which are resuspended in 300μL of sample buffer.

2. 2D-ELECTOPHORESIS
Material
Protean IEF system
Cup loading tray set
Protean IEF cell
Protean II XI basic unit, casting stand
Protean II XI cell IPG conv. kit
IPG ready strip pH 3-10 7 cm
IPG ready strip pH 3-10 17 cm
100x Bio-Lyte 3-10 Ampholyte, 1 mL

Sample buffer (sb)

Tris-HCl 40mM, pH 7.4
Urea 8M
Thiourea 7M
Tributhylphosphine (TBP) 2mM
CHAPS 4%
Ampholytes 0.2%

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Bromophenol blue 0.0002%

Sample preparation
Samples must always be dissolved before running in 2D-electroforesis,
independently of the previous treatment. Cationic detergents must be avoided
and low-salt buffers must be used to interfere as little as possible with the
focusing of the sample in the first dimension.

Starting material:
a. Precipitated proteins: dilute in 300μL of SB*
b. Brain sample:
b.1. Sample homogenization in SB without bromophenol blue and
without ampholytes. The ratio sample: SB described for the
precipitation protocol is mantained.
b.2. Centrifuge 12,000 rpm for 15 min at 4ºC. (Some proteins may be
lost during this step. The speed of the centrifuge must be adapted
depending on the localization (nuclear or cytosolic) of the studied
proteins).
b.3. Discard the pellet.
b.4. The amount of protein from the supernatant is quantified with the
Bradford method, using BSA as a standard. * (The amount of protein
to be loaded in each strip depends on the length of the strip, with 300
μg of protein as a minimum, in 300μL of final volume onto 17cm IPG
strips).

First measurement
To avoid contamination during the final detection steps, all the material must
be kept clean with dd H2O, and all the steps must be performed wearing
gloves. After dissolving the sample the first measurement must be performed.
Acrylamide strips with distributed ampholytes (IPG strips) are used depending
on the pI of the proteins to be separated. After distributing the protein sample
all along the IPG strip and following incubation at room temperature for 1
hour (passive hydration), the focus is programmed in the IEF cell as follows:
active hydration at 50V for 16 hours; 300V for 2h; 500V for 2h; 1000V for 2h;
8000V for 8h; and, finally, 8000V for 10h. Running conditions will be adapted

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depending on the proteins required for study. IPG strips can be kept at –20ºC .
The running of the second measurement does not need to be carried out
immediately.

Second measurement
IPG Strips equilibration

Equilibration buffer (eb).

(Can be prepared and kept at –20ºC)
Urea 6M
SDS 20%
Tris/HCl 0.05M, pH 8.8
Glycerol 20%
dd water
IPG strips must be equilibrated before starting the second measurement.
The equilibration is performed by a 2-step incubation protocol.
1. 10 minutes incubation in EB plus dithiothreitol (DTT) 2% added
just before being used.
2. 10 minutes incubation in EB plus iodoacetamide 2.5% instead of
DTT.
After this the IPG strips are soaked in running buffer and loaded onto SDS-
PAGE electrophoresis gel, the acrylamide percentage of which will depend on
the molecular weight of the proteins to be detected. The best approach is to
perform a gradient gel (8-15% acrylamide) to cover as many proteins as
possible for a first screening, and then, depending on the results, the gradient
and/or the acrylamide percentage can be adjusted depending on the proteins
required for study.

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DETECTION METHODS AFTER SECOND-DIMENSION
ELECTROPHORESIS
After second dimension electrophoresis, acrylamide gels are treated differently
depending on the method of protein detection.
• For oxidized protein detection: Western blot against anti-DNP antibody.
• For a general screening of the electrophoresis: Comassie blue staining.
• For a more accurate analysis of the samples to detect small differences
between control and pathological samples, and for sequencing protein
spots: Silver Staining or Cypro Ruby fluorostaining.

MASS SPECTROMETRY COMPATIBLE SILVER STAIN PROTOCOL

• Described amounts are for a 17 cm gel.
• All reagents need to be prepared in dd H2O and the protocol must be
followed using glass containers.
• Procedure described following the instructions of the manufacturer: Silver
Staining Kit Protein (Amersham Biosciences)

Fixing: 15 min x 2
- 40% MetOH: 100 mL
- 10% Acetic acid: 25 mL
- dd H2O: make up to 125 mL
This step can be performed O/N

Sensitizing: 30 min.
- 30% MetOH: 37.5 mL
- 5mL Sodium Thiosulphate (5% w/v)
- 8.5g Sodium Acetate
- dd H2O: make up to 125 mL
5 X 5 min washes with dd H2O (this step prevents background precipitation
while silver reaction is added)

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Silver reaction: 20 min BrainNet Europe II
Project Co-ordinator:
- 12.5 mL silver solution (2.5% w/v)
Prof. Dr. Hans Kretzschmar
- dd H2O: make up to 125 mL
Contact:
2 X 1 min washes in dd H2O
www.brainnet-europe.org

Developing: incubation time depends on the amount of gel.

- 3.12 g sodium carbonate Network of Excellence funded by the
EC in the 6th Framework Program
- 50 μL formaldehyde (37% w/v)
“Life Science”
- dd H2O: make up to 125 mL

Stopping: 10 min
- EDTA-Na*2H2O (3.65g)
- dd H2O: make up to 125 mL
3 X 5 min washes in dd H2O

Copyright:
© by author(s) 01.01.2008
Reproducing, redistributing, or making
commercial use of this information is
expected to adhere to the terms and
conditions asserted by the copyright
holder.
All rights reserved
Edited: 28.02.2008
Filename Protein Preservation

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