3.4.4 Dot Blot 1. Prepare the filter paper and the nitrocellulose membrane for
dot botting by equalizing both in PBS for 5 min.
- Samples should be stored on ice till finale usage.
- Vortex samples thoroughly before usage.
- Apply 200μL of PBS to each dot to purge the system. Make
sure that the solutions are gone before moving to the next step
(seeNote 10). - Apply 100μL of the samples slowly in the center of the grid.
Avoid air bubbles which are preventing the normal flow-
through (seeNote 11). - Apply 200μL of PBS to each dot again to purge the system.
Make sure that the solutions are gone before moving to the
next step. - Let the lift pump work for another 10 min.
- Remove the filter paper and let the nitrocellulose membrane
dry for 60 min at 60C. - Block the membrane with PBSþ5% milk for 1 h at room
temperature. - Incubate the antibody in PBSþ5% milk overnight at 4C.
Antibody concentration 1μg/mL. - Wash the membrane five times for 5 min at room temperature
with PBS-T. - Incubate the secondary antibody according to the manufac-
turer’s protocols in PBS þ 5% milk for 2 h at room
temperature. - Wash the membrane five times for 5 min at room temperature
with PBS-T. - Detect the membrane with chemiluminescent detection
reagents of choice.
Fig. 4Tyrosine nitration as a measure of peroxynitrite formation (dot blot). Dot
blot signals of bovine serum albumin samples without pretreatment (negative
control) and with peroxynitrite (100 mM) preincubation as a positive control504 Swenja Kro ̈ller-Scho ̈n et al.