Transfer proteins to the membrane for Western blot analysis

Transfer proteins to the membrane for Western blot analysis


Once proteins are separated by
gel electrophoresis, most Western blot workflows
require transferring the separated proteins from the
polyacrylamide gel to a membrane before proteins of
interest can be detected. The most effective transfer
method is electroblotting, in which an electric field is
applied perpendicular to the plane of
the gel, causing proteins to migrate to a
protein-binding membrane. Electroblotting can be performed using wet and semi-dry transfer
systems. Although each system has unique
features, they both require building a
transfer stack or “sandwich” to ensure that the
gel and membrane are pressed closely
together. The transfer stack includes the
gel, a charged membrane such as
nitrocellulose or PVDF and several pieces of filter
paper. Western blot transfer also
relies on an electrically conductive buffer
that often contains methanol or another solvent to
promote protein binding and to help remove SDS from the
gel. Before building the transfer
stack, the gel, membrane and filter
paper are equilibrated for up to 15 minutes in fresh
transfer buffer. Also, if PVDF is used, the
membrane should be pre-wetted in 100% methanol or another
solvent before equilibrating. Electric current flows from the
cathode, which is color-coded
black, to the anode, which is red, and
therefore the transfer stack must be built so that the
proteins in the gel will flow with the current and attach
to the membrane. When assembling and orienting an
electroblot, it is helpful to remember that
the current and proteins “runs
to red”, that is, toward the red
electrode. Let’s build a transfer stack for
wet electroblotting, in which the transfer sandwich
is submerged in a tank of transfer buffer through which
the electric current passes. Wet transfer systems often come
with sponges and color-coded cassettes that hold
the stack together during
transfer. Place a sponge that has been
pre-wetted with transfer buffer onto the red side of the
blotting cassette. Place a piece of filter paper on
top of the sponge, pipette transfer buffer onto the
filter, and then gently roll the membrane onto
the filter to minimize air bubbles that
form between the 2 layers. After pipetting more transfer
buffer onto the membrane, stack the gel onto the membrane. Handle the gel with care so that
it doesn’t stretch or tear when placed onto the
filter paper. If the gel is damaged or
stretched, pipette more transfer
buffer between the gel and filter paper
layers to help you repair or
adjust the gel. Place another piece of filter
paper on top of the membrane, and then roll the stack out to
remove any air bubbles that can impede protein
transfer. Finally, place another wet
sponge on top of the transfer
stack, and clamp the cassette shut. Then place the cassette
vertically in a transfer tank, taking care that the colored
sides of the cassette are correctly oriented to the
color-coded anode and cathode. Fill the tank with transfer
buffer, cover it with the lid and connect it to a power
source, making sure that the correct cables are connected to
the anode and cathode. Then apply an electric field at
the appropriate current or
voltage for 1 hour to overnight,
depending on the sample, the number of transfer
sandwiches in the tank and the equipment used. Make sure that the transfer tank
is cooled by performing the transfer in a
cold room, inserting ice packs
into the tank or using a transfer system with
an integrated cooling base to dissipate the heat produced by
the electric field. After the transfer has been
completed, turn the power off and unplug the tank from the
power source. Remove the cassette from the
tank and gently open it to
reveal the transfer sandwich. Now let’s build a transfer stack
for semi-dry electroblotting. Instead of clamping the sandwich
between sponges in a cassette and submerging it in a tank,
semi-dry blotting systems place the sandwich directly in
contact with the anode and
cathode and use thick filter layers as
the reservoir for transfer
buffer. In this demonstration, the
sandwich is built on the anode plate of a semi-dry
blotter. First, place several pieces of
pre-soaked filter paper onto the anode. Then, because the electric field
runs to red, the anode, place the pre-wetted membrane
onto the filter paper followed
by the gel. Top the stack with another thick
layer of pre-soaked filter
paper, and roll the stack to remove any
air bubbles. Place the cathode plate on top
of the sandwich and activate the electric field. After the transfer is completed,
open the blotter to reveal the
transfer stack. Once the transfer is completed
using either wet or semi-dry
transfer methods, remove the membrane from the
sandwich and proceed to blocking
and probing to detect your proteins of
interest. Membranes can rapidly dry out,
though, so keep the membrane
hydrated between each step in the Western
blot procedure.