Bio-Rad Immun-Star™ AP Chemiluminescence Kits - Manual (Page 19)

c. Dot-blotting – cut the membrane sheet to the
appropriate size. Draw a grid on the membrane with a
pencil. Wet the dry membrane by slowly sliding the
membrane at a 45° angle into TBS. (PVDF membranes
must first be wetted in methanol; consult membrane
instructions for complete information.) Remove the
thoroughly wetted membrane from the TBS and dry it
on filter paper for approximately 5 min. Apply antigen
sample to each grid square using a syringe or a
variable pipet, by displacing a 1 µl drop of sample to
the tip of the syringe or pipet and gently touching it to
the surface of the membrane. If the sample is very
dilute, it is possible to apply successive 1 µl doses at
the same spot by letting the previous sample applica-
tion dry completely before adding an additional dose.
In all cases, the membrane should be allowed to dry
completely before proceeding to the blocking step. For
PVDF following this drying step, rewet with 50%
methanol before transferring to the TBS solution in step
2 below.
2. Wash – following transfer, wash the membrane in TBS for
5–10 min with gentle agitation at room temperature (RT).
Decant the wash solution and repeat the wash step one
more time. These washes are important to reduce spotted
or blotchy background problems.
3. Blocking step – after the antigen is applied, using one of
the above methods, immerse the membrane, at a
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