Normalize volume of all samples and place on ice. A minimum
volume of 200 μL is recommended.
Assemble Bio-Dot SF apparatus as described in the product
manual.
Soak 3× Bio-Dot SF fi lter paper in wetting buffer for 10 min.
Cut a 9 × 12 cm sized piece of 45 μm PVDF membrane and
place in 100 % (v/v) methanol for 2 min.
After activation, discard methanol and rinse membrane with
water. Transfer the membrane to wetting buffer.
Place the fi lter papers above the support plate followed by the
activated membrane on top of the fi lter paper.
Place the sample template on top of the membrane and tighten
the screws on the sample template.
Attach the apparatus to the vacuum source. Set the valve in the
apparatus to atmosphere setting. Ensure that the vacuum is
not turned on.
To equilibrate the wells, add 100 μL of buffer B to all wells and
turn on vacuum for 30 s to fi lter the buffer through. Ensure that
the membrane does not dry out during fi ltration ( see Note 7 ).
Turn off vacuum and discard any leftover liquid in the wells.
Load the samples on the wells starting with the most dilute for
each set (in the order of D4, D3, D2, and then D1). Fill all
unused wells with buffer (same volume as sample) and seal
them off using Scotch Tape.
Let sample stand in the wells for 10 min at room temperature
to allow for protein binding to the membrane.
After incubation, fi lter the sample through by turning on the
vacuum source ( see Note 8 ).
Seal off wells with Scotch Tape as soon as the sample has fi l-
tered through to prevent drying ( see Note 9 ).
Add 200 μL of buffer B containing 0.5 % (v/v) protease inhib-
itors to each sample well and let stand for 2 min at room
temperature.
Repeat the fi ltration process as described in steps 18 and 19.
After fi ltration, leave the vacuum on and unscrew the sample
template.
Turn off the vacuum, remove the membrane and let it air-dry
completely.
Activate the membrane in methanol as described in steps 9
and 10.
Place the membrane into Coomassie Blue R-250 solution and
stain for 15 min on a rocking platform.
