Dot Blot (Western)

1. Cut membrane (Nitrocelluose) to size of 96 well apparatus.
2. Prewet with 1X TBS, place on apparatus.
3. Tighten in cross pattern, vacuum for a moment (until TBS just disappears), then tighten again.
4. Add 100µl of samples.
5. Let samples sit for 30 min. with stop cock is turned to air.
6. Vacuum off remainder of liquid. If some wells will not get empty, help by stirring with a micropipette tip.
7. Add 50µl of TBS to each well to rinse membrane within the apparatus.
8. Take membrane out of the apparatus, rinse once in 1X TBS.
9. Block nonspecific binding with 5% nonfat dry milk TTBS for 90 min. on shaker. Incubation O/N at 4°C not necessarily on a shaker is also possible.
10. Follow the protocol of western blotting (primary and secondary antibody, ECL detection system).

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