Team:Freiburg/Protocols/Dot Blot
Dot blot
Dot blot
material: same buffer as for western blot Western Blot towbin et al.
duration: 4-5h or o/n
In a dot blot the biomolecules to be detected are not first separated by electrophoresis. Instead, a mixture containing the molecule to be detected is applied directly on a membrane as a dot.
- activate the PVDF membrane with MetOH
- rinse the membrane with 1x blotting buffer see Western Blot towbin et al.
- mark with a pen directly on the membrane your spotting pattern
- pipette 1-2µl of you sample on the membrane
- let air dry the spots for 20-30min, RT
- incubate the membrane with blocking buffer (1h, RT)
- perform 3×10 min the washing step (1xTBS-T)
- incubate the membrane with 1st antibody for 1h-2h, RT
- wash the membrane 3x 10min with 1xTBS-T
- incubate with enzyme-labeled secondary antibody, 1h,RT or o/n, 4°C
- wash membrane 3x 10min with 1x TBS-T
- detect with chemiluminescent imaging system
