supplies, and equipment:
- Tris-Glycine electrophoresis buffer:
0.25 M Tris
1.92 M glycine
15.1 g Tirs
72 g glycine
50 ml 10% SDS
q.s. 1 L H2O
- 1X Electrophoresis buffer - prepare fresh
100 ml 5X stock
400 ml DI H2O
- 4X SDS gel-loading buffer w/o DTT (aliquots stored at
25 mM Tris-HCl (pH 6.8 at 25°C)
8% w/v SDS
0.04% w/v bromophenol blue or phenol red
For 100 ml:
Add 25 ml 1M Tris HCl to 40ml glycerol and stir
While stirring, add 8 g SDS until clear
Add 10 ml 0.4% bromophenol blue (0.4% w/v bromophenol blue stir O/N
q.s. with DI H2O
- 10X transfer buffer w/o methanol (RT)
30.3 g (0.25 M) Tris base
144 g (1.92 M) glycine
q.s. 1 L DDI H2O
- 1X transfer buffer w/ methanol (may be used twice; store at 4°C
100 ml 10X transfer buffer
800 ml DIH2O
200 ml methanol
- 10X TBS: (stable at 4°C for several
24.2 g Tris base (100mM)
80 g NaCl (0.9%)
Adjust pH to 7.6 with HCl
q.s. 1 L with DDI H2O
(Add 1 ml Tween-20 to 1 L 1xTBS as working solution store at 4°C
- Blocking buffer: 1X TBS, 0.1% Tween-20 with 5% w/v nonfat dry
Mix 10ml 10X TBS in 90 ml water
Add 7.5 g nonfat dry milk (Bio-Radad: #170-6404) and mix
While stirring, add 0.15 ml Tween-20
- Antibody dilution buffer: X TBS, 0.1% Tween-20 with 5% BSA
(For some antibodies indicated in vendor manual)
Mix 2 ml 10X TBS to 18 ml water
Add 1.0 g BSA and mix well
While stirring, add 20 µl Tween-20 (100%)
- Ponceau S solution (Sigma #P7170)
- DTT reducing buffer (Invitrogen #NP0004)
- Pre-stain dual color protein standard (Bio-Rad: #161-0374,
store at -20°C)
- Biotin linked ladder detection pack (Cell signal: #7727 store
Gel: 4-12% Tris-HCL Ready Gel 10 well
Apparatus: Bio-Rad MP3 (Run at 100V 1h 15
- Extra-thin loading pipette tips
- Ice bucket
- PCR tubes
- 3MM Whatman paper
- Trans-Blot transfer medium pure nitrocellulose membrane 0.45
µm. (Biorad: #162-0145 )
- Flat head forceps
- Plastic tank
- Western lightening chemiluminescence reagent plus (Pierce
- Film (Kodak, #NEF586 Blue XB-1)
- Restore Western blot stripping buffer (Pierce #021059)
- PCR machine and
- Prepare 500 ml 1X electrophoresis buffer in H2O and
ice bucket. Turn on PCR machine to pre-warm.
- Aliquot protein samples to PCR tubes (~25 μl/sample), add 1/3
volume of loading buffer and 1/10 volume of DTT; mix by pipetting
up and down. Keep on ice! Include a negative control
(if necessary) and two protein ladders (pre-stained and biotin
- Heat the samples and the biotin linked protein ladder to 95 -
100°C in PCR machine for 5 min. Do NOT heat the prestained
- Immediately remove tubes from the PCR machine and place on ice
for 3 min. Caution - This is HOT! Place gloved hands
over the lids of tubes to prevent them from popping off.
- Microcentrifuge samples at 12,000 rpm, at 4°C for 5
- Set up the gel and tank system while the centrifuge is
spinning. (Slowly remove comb from gel).
- Slowly load 10 - 20 µl of sample onto gel using extra thin
- Run at 100 V for 1.5 hr or until the loading dye reaches the
end of the gel.
- While the gel is running, prepare the prewet membrane as
a. Prepare 1X transfer buffer
b. Remove the precut nitrocellulose membrane and cut 6 sheets
(3 for each side) of 3 MM Whatman filter paper to the same size as
the precut membrane (usually 3" x 4").
c. Soak the transfer membrane, filter paper, and sponges in
the transfer buffer at least 30 min before gel is finished.
There are 2 sponges, 6 pieces of filter paper, and 1 nitrocellulose
membrane per gel. Use forceps to manipulate everything into
the transfer buffer.
- Just prior to completion of the gel run, prepare a plastic box
by pouring in enough 1X transfer buffer to cover gel.
- When gel is finished:
a. Cut a corner on the bottom right of gel. Also remove
the top wells.
b. Transfer gel to plastic box and soak gel in transfer
buffer for 30 min. Keep transfer buffer on ice!
- Place the tank for transfer in a plastic tub and pack with
- Take transfer apparatus to the cold room and perform the
a. Pour all of available transfer buffer into the tank
(including all the buffer used previously).
b. Assemble "sandwich" for Bio-Rad Transblot using the porous
plastic sandwich apparatus. (membrane size should be 1 - 2 mm
larger than gel). The black part of the sandwich apparatus
faces the cathode (negative) and the white part faces the anode
c. Assemble the "sandwich" in the following order: black side
of porous plastic sandwich, sponge, 3 pieces of 3MM Whatman filter
paper, gel, membrane, 3 pieces of 3MM Whatman filter paper, sponge
and white side of porous plastic sandwich. Use forceps to
handle everything except the gel. DO NOT LET MEMBRANE
d. RRemove bubbles between the sandwich and clamp the gel
e. Place gel inside the red and black apparatus; black side
of the sandwich facing the black part of the apparatus.
f. Cover the lid and immerse the entire unit in ice, burying
- Transfer the Mini-Transblot at 100 V for 1 hr.
- While transfer is running, prepare 1X TBS with Tween-20 (This
buffer may be used multiple times).
- Upon completion cut the membrane at the same corner as the gel
corner cut and rinse the membrane with DI H2O.
- Wash membrane in H2O by shaking for 5 min.
- Pour Ponceau Stain onto membrane:
a. Stain for 1 min. Pour off the stain (return to tube)
and rinse several times with DI H2O until protein bands
b. According to the size of the protein, mark the place you
want to split the membrane. Cut the membrane in sections that
contain the specific proteins.
c. Cut the bottom right corners of the membrane sections and
place them carefully in size matched boxes (small to small, large
to large) containing TBS.
- Wash with 1X TBS 3 times for 5 min and shake at RT.
- Incubate in blocking buffer for 1 hr at RT, shaking at 50
- While blocking, prepare the primary antibody at appropriate
dilutions in blocking buffer (i.e. GAP-DH at 1:8,000 ).
- Incubate membrane in appropriate volume of primary antibody +
buffer; gently agitate at 50 rpm O/N, at 4°C (cold room).
- The next day, wash 3X for 10 min each with TBS/T.
- While washing, prepare secondary antibodies. Prepare
HRP-conjugated secondary antibody (1:10,000, specific antigen
detection) in appropriate volume of blocking solution the as
well as HRP-conjugated anti-biotin antibody (1:6,000, detection of
biotinylated protein marker).
- Incubate membrane by gently shaking at RT for 1 hr with
respective secondary antibody.
- Wash 3X for 10 min each with TBS/T.
- While washing, prepare for chemiluminescence as follows:
a. Open the film cassette.
b. Lay a large piece of plastic wrap on top of the bottom of
the intensifying screens and smooth to remove wrinkles.
c. Mix 1 ml oxidizing reagent with 1 ml luminol reagent.
- Using forceps, arrange the membranes (protein side up) on the
plastic wrap (note order).
- Pipette 2 ml of the mixed reagents onto the membrane
- Fold the plastic wrap over the membrane to seal and close the
- Take the following to the dark room: scissors, forceps, film in
black bag, film cassette, pen, and timer.
- To develop:
a. Open the film cassette and place film on top of the
plastic wrapped membrane.
b. Conduct multiple exposures ranging between 2 sec to 20
min, depending on the strength of the signals. (Cut film
corner for orientation).
d. Develop film (Small pieces of film will jam the machine; 8
X 10 cm is the minimum size).
- After development, rinse membrane 2 - 3 times in fresh PBS or
- To strip and re-blot:
a. Wash membrane in Western blotting stripping
solution, RT for 20 min.
b. Perform three 5 min washes in 1X TBS.
c. If necessary, re-stain with Ponceau beginning with step 22
or 23, depending on antibody species.
Written by Yanyun Gu, Sara Chen 7/21/08