WESTERN BLOT INSTRUCTIONS
PART I
1. Gel cassette preparation
 See figure 1 to 4 on the Mini-PROTEAN® 3 Cell Assembly Guide.
 Pour water to test its water tightness.
2. Gel Casting
 Take out the water and insert a piece of filter paper to dry the area in between the glass plates.
 Place a comb completely into the assembled gel cassette.
 Mark the glass plate 1 cm below the comb teeth. This is the level to which the running gel is
poured. Remove the comb.
 Prepare the running gel.
RUNNING GEL (for 2 gels)
Reagents
Acryl bis 30% mL)
Running buffer (mL)
SDS 10% (µL)
H2O (mL)
APS 10% (µL)
Temed (µL)
6%
3
3.8
150
8
300
20
8%
5.32
5
200
9.4
200
20
10%
5
3.75
150
6
150
20
12%
6
3.75
150
5
150
20
15%
7.43
3.75
150
4
150
20
 Pour the gel (~ 6.5 to 7 mL per cassette) smoothly to the mark to prevent it from mixing with
air. Immediately overlay the running gel with water.
 Allow the gel to polymerize for 15 to 30 minutes. Take out the water and insert a piece of filter
paper to dry the area in between the glass plates without touching the gel!!!
** At this point, the running gel can be stored at room temperature overnight. Add 5 mL of a 1:4
dilution of 1.5 M Tris-HCl, pH 8.8 buffer to the running gel to keep it hydrated.**
 Prepare the stacking gel.
STACKING GEL (for 2 gels)
Reagents
Acryl bis 30% mL)
Stacking buffer (mL)
SDS 10% (µL)
H2O (mL)
APS 10% (µL)
Temed (µL)
4%
0.665
1.25
50
3
25
5
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 Pour the gel smoothly to avoid bubbles between the glass plates. Continue to pour until the top
of the short plate is reached.
 Insert the desired comb between the spacers starting at the top of the spacer plate, making sure
that the tabs at the ends of each comb are guided between the spacers.
 It is easiest to insert the comb starting at an angle and insert well 1 first, then 2, 3, and so on
until the comb is completely inserted.
 Seat the comb in the gel cassette by aligning the comb ridge with the top of the short plate.
 Allow the stacking gel to polymerize for 15 to 30 minutes.
3. Final preparation of the samples (cell lysates and supernatants)
 Thaw cell lysates and supernatants on ice.
** For cell lysates, the quantity of protein to load will depend of the importance of the protein
expression and of the antibody sensitivity.**
 Cell lysates are diluted with Laemmli buffer (blue) 5X (kept at 4oC, in a box, lab 216, on
Severine shelf) for a 1:5 dilution (example : if you want to load 20 µg of proteins and the
concentration of the cell lysate is of 5µg/µL → you need to take 4 µL (cell lysate) + 2 µL
(loading dye 5X) + 4µL (loading dye 1X) = 10 µL total volume of sample).
 Keep the same final volume among samples to ensure homogeneous migration.
 Vortex and do a quick spin.
 Just before loading, boil for 5 minutes the cell lysates and supernatants samples, and also an
aliquot of loading dye 1X.
** Do not place samples back on ice after boiling to avoid SDS precipitation.**
 Vortex samples and do a quick spin.
 Once the stacking gel polymerize, gently remove the comb and rinse the wells thoroughly with
distilled water.
4. Mini-PROTEAN 3 Electrophoresis Module Assembly
 See figure 1 to 5 on the Mini-PROTEAN 3 Electrophoresis Module Assembly Guide.
 Fill the inner chamber with tank buffer 1X.
5. Samples loading
 Load the samples into the wells with gel loading tips.
 If using Bio-Rad’s patented sample loading guide, place it between the two gels in the electrode
assembly : use the sample loading guide to locate the sample wells.
 Insert the pipette tip into the slots of the guide and fill the corresponding wells with either the
protein ladder (5 µL) (at -20oC, lab 216, box Isao WB), the samples (15 to 60 µL), or the
loading dye 1X (5 µL) (into the empty wells to obtain a linear migration front).
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** Load the samples slowly to allow them to settle evenly on the bottom of the well. Be careful not
to puncture the bottom of the well with the pipette tip.**
 Refill the inner chamber (if necessary) and the outer chamber with tank buffer 1X.
 Place the lid on the Mini Tank making sure to align the color coded banana plugs and jacks (red
with red, and black with black).
 The correct orientation is made by matching the jacks on the lid with the banana plugs on the
electrode assembly.
 Insert the electrical leads into a suitable power supply with the proper polarity.
 Apply power to the Mini-PROTEAN 3 cell and begin electrophoresis.
 To begin with, adjust power to 60V to let the samples migrate until the running gel. Then,
increase the voltage to 110-120V for 1h to 1h30 (check the migration front).
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WESTERN BLOT INSTRUCTIONS
PART II
1. Gel removal
 After the electrophoresis is complete, turn off the power supply and disconnect the electrical
leads.
 Remove the tank lid and carefully lift out the inner chamber assembly.
 Pour off and discard the tank buffer.
** Always pour off the buffer before opening the cams to avoid spilling the buffer.**
 Remove the gel from the gel cassette sandwich by gently separating the two plates of the gel
cassette. The green, wedgeshaped, plastic gel releaser may be used to help pry the glass plates
apart.
 Cut the stacking gel.
 Cut 1 or 2 corner(s) of the gel (nearest to the protein ladder if protein ladder was loaded into 1
well only) for identification (example : gel A → 1 corner cut and gel B → 2 corners cut).
 To remove the gel, first slice the tape along the sides of the gel cassette where the inner glass
plate meets the outer plastic plate.
 Put the gel into the transfer buffer 1X while awaiting to make sandwich.
2. Transfer on membrane
 Cut the membrane (6 x 9 cm)(also cut the corner(s) accordingly to the one(s) cut on the gel) and
the filter paper (8 x 10 cm).
** Always wear gloves when handling membranes to prevent contamination.**
 If using PVDF (Polyvinylidene Difluoride) membrane, wash the membrane into 100%
methanol for 30 seconds and then 2 x 5 minutes into water (or until the membrane sinks in the
water).
 Equilibrate the gel, the membrane, the filter paper and the fiber pads in the transfer buffer 1X
for 15 minutes to 1 hour (depending on the gel thickness).
 Prepare the sandwich:
Place the cassette with the black side down
Place 1 fiber pad on the black side of the cassette
Place 1 sheet of filter paper on the fiber pad
Place the gel on the filter paper
Place the membrane on the gel (corner(s) of the membrane on corner(s) of the gel)
Place 1 sheet of filter paper on the membrane
** Use a glass tube to gently roll air bubbles out **
Place 1 fiber pad on the filter paper
Close the cassette firmly and lock it closed with the white latch
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Place the cassette in the module → black against black and transparent against red.
Repeat for the other cassette.
Put the module in the tank with a magnet under the module.
Add the frozen Bio-Ice cooling unit into the tank next to the black side module.
Completely fill the tank with transfer buffer 1X.
Put on the lid. Plug the cables into the power supply and run the blot for either 1h at 4 oC at
100V OR overnight in the cold room at 30V, with the magnet stirring .
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WESTERN BLOT INSTRUCTIONS
PART III
1. Labelling
** While opening the sandwich to take out the membrane, do not forget to notice on which side of the
membrane the proteins were transferred.**
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Rinse the membrane with T-TBS, 2 x 5 minutes.
Saturate the membrane in a blocking solution with milk for 1 to 2 h.
Rinse the membrane quickly for 5 minutes with T-TBS.
Incubate the membrane with the primary antibody in T-TBS for 1 h at room temperature OR
overnight in the cold room.
** Recuperate the antibody and store it at -20oC.**
 Rinse the membrane with T-TBS, 3 x 10 minutes.
 Incubate the membrane with the appropriate secondary antibody (example : if using mouse/antihuman GATA-3 monoclonal primary antibody, then use ECL-sheep/anti-mouse-HRP
monoclonal secondary antibody) in T-TBS with milk for 1h at room temperature.
** Recuperate the antibody and store it at -20oC .**
 Rinse the membrane with T-TBS, 4 x 15 minutes.
2. Verification of the transfer
 In the meantime, verify the efficiency of the transfer by incubating the gel in the Coomassie
blue for 1h.
 Then, rinse the gel in a decoloration solution for 10 minutes and incubate the gel in the same
solution overnight (put a piece of towel paper next to the gel to absorb the coloration).
 The next day, if necessary, change the solution and the towel paper, and incubate the gel further
more.
3. Developing
 Remove excess washing buffer from the membrane (decant the buffer by gently touching one
side of the membrane onto an absorbent paper) and lay down flat on saran wrap.
** Make sure that the side of the membrane with the transferred proteins is facing up.**
 Soak the membrane in E.C.L. Plus (1 mL of solution A and 25 µL solution B or 0.06 mL of
E.C.L. /cm2 of membrane surface) for 5 minutes at room temperature in the dark.
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 Remove the excess reactant with an absorbent paper and wrap the membrane between 2 acetate
sheets.
For acquisition with the fluorochem program:
 1 minute HIGH/LOW, to check the presence of bands.
 50 minutes NORMAL/HIGH, for acquisition.
For development of film:
 Turn off the lights and only leave the red light on.
 Prepare enough of “developer” 1X (from the 5X, found under the sink) with water ***Sensitive
to light*** and also prepare enough of “fixative” 1X (from the 5X, found under the sink) with
water.
 Prepare 3 pyrex with the “developer”, H2O, and the “fixative”.
 After exposing the membrane to the film for a certain amount of time, completely submerge the
film into the “developer” (20 to 30 seconds), H2O (20 to 30 seconds), and the “fixative” (20 to
30 seconds).
*** The size of the film must depend of the amount/size of the membrane(s) and the time of
exposure will depend of various factors (Ab, protein, amount of sample, …)***
 Wash the film under tab water and observe the presence or not of bands.
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