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[hurchu]

I have been having trouble with the dot blot. I was hoping to see the DNA methylation difference in different strains. And hybridize the blot with genomic DNA with anti-methyl-C-antibody and then with secondary goat-anti-mouse hrp antibody and used west pico chemiluscent kit for signal detection.

What I got is reverse image! The background are totally dark and the dots are pale. Could anyone help me with this?

I have looked at chemislucent kit manual and it tells reverse image tells too much hrp in the system. The problem is that I have used the same concentration and it was fine for twice. And then everything went wrong. Can there be any other possibility?
Another question, if it is indeed too much hrp in the systerm, how can I strip it and use the same blot again? is 0.4M NaOH a good option?

Attached is the procedure starting from blocking. Thanks a lot.

10. Block membrane with 1x PBS-T + 5% non-fat dietary milk (25ml, an hour).
11. Wash 3 x for 5 min in 1 x PBS-T.
12. Incubate O/N at 4C in 1xPBS-T (25ml with 2.0uL of monoclonal mouse a-5meC antibody)
13. Next day, wash 3 x w/PBS-T for 5 min.
14. Briefly wash 2x in TBS
15. Incubate with Secondary antibody (2.0uL of goat-a-mouse HRP antibody) an hour in PBS-T.
16. The blot was subsequently washed 3x in 1 × PBS-T for 5 min each and finally rinsed with distilled water.
17. Prepare working detection solution by mixing equal parts of the stable peroxide solution and the luminal/enhancer solution). Incubate blots 5 minutes with working solution (Super signal west pico chemiluscent kit). Use 0.1ml working solution per cm2 of membrane. Caution: light emission is intense. Any movement between the film and membrane can cause artifacts on the film.
18. develop film.