Western Blot (Tank Transfer)

A. Transferring
1. Prepare the transfer buffer in amounts that will suffice for gel
equilibration, sandwich assembly, and electrophoresis.

2. Rinse gels briefly in ddH2O and equilibrate for 15 min in transfer

buffer.

3. For each gel, cut one piece of membrane and four pieces of filter
paper, and four pieces of soft padded sponges to the dimensions of
the gel.
4. If using PVDF membranes, activate membranes in methanol for 30
seconds.
5. Equilibrate the membranes, in transfer buffer for 15 minutes.
6. Soak soft padded sponges and filter papers in transfer buffer.
7. Place 2 soft padded sponges on top of the black side of the
cassette, submerged in buffer and push on the fiber pad with gloved
fingertips to thoroughly wet the pad.
8. Place 2 pieces of filter paper on top of the soft padded sponges (it
should be wetted immediately).
9. Gently place the preequilibrated gel on top of the filter paper, run a
wet, gloved finger across the gel to remove any air bubbles that
may be trapped underneath the gel.
10.Carefully place the preequilibrated membrane on top of the gel and
make sure the membrane is correctly positioned as it touches the
gel, then use the roller to remove any air bubbles and to ensure
proper contact between the gel and membrane.
11.Wet 2 pieces of filter paper in transfer buffer and place it on top of
the membrane.
12.Place 2 soft padded sponges on top of the filter papers.
13.Use the roller to remove any air bubbles and to ensure proper
contact between the gel and membrane.

14.Once the cassette is closed and locked, insert it into the tank with
the latch side up. Make sure the black cassette plate faces the black
electrode plate.
15.Add transfer buffer to the tank until the buffer level reaches the fill
line.
16.Place lid and run at 35mA for 1 to 2 hours

B. Blocking
1. Prepare 5% Blotto:

2.5g Blocking Grade Blocker (Bio-Rad non-fat milk) + 50ml 0.1%

PBS-T

2. Place membrane in container containing blocker.

3. Incubate for 1 hour at RT or overnight at 4C with shaking

C. Primary Antibody Incubation
1. Prepare Primary Antibody (anti sample host) using 5% Blotto as
diluent.

2. Place membrane in container containing primary antibody.

3. Incubate for 1 hour at RT or overnight at 4C with shaking
D. Secondary Antibody Incubation
1. Prepare Secondary Antibody (anti primary body host) using 5%
Blotto as diluent.

2. Wash blocked membrane 3x with 0.1% PBS-T

3. Place membrane in container containing secondary antibody.

4. Incubate for 1 hour at RT with shaking
E. Detection

1. Prepare chemiluminescence HRP substrate (Milipore Luminata

Classico Substrate)

2. Wash blocked membrane 2x with 0.1% PBS-T and 1x with PBS

3. Cover membrane with 1ml HRP substrate and remove excess

4. Expose in SynGene reader as per desired time.