Did you try apply onto pvdf membrane? maybe your sample sensitive with nitrocellulose or maybe lost during blocking step

Hello

look at the paper in link which used this protocol  " cells growing in log phase was resolved by SDS–PAGE, electroblotted onto nitrocellulose membranes and blocked for 2 h at room temperature (RT) in 5% non-fat dry milk. Membranes were incubated with anti-DNMT1 (Abcam, Cambridge, UK: ab54759) overnight at 4°C, followed by HRP-conjugated secondary antibody for 2 h at RT before detection using ECL (Amersham, Amersham, UK) "

Good Luck

http://hmg.oxfordjournals.org/content/20/16/3241.full

Dear Salem, thank you very much for forwarding this paper, it's very helpful. 

Mohammed, yes I did the blocking step, initially with BSA but now I am repeating it in milk.

Atirah, I tried with PVDF but it idid not work at all and my lab works mainly with nitrocellulose so I am keen to persevere with nitrocellulose...

Thank you all.

Dear Pavlina,

Ok then, maybe you can emphasize about time consuming when applied secondary antibodies (not longer than 1 hour). and might be your protein in sensitive in high temperature, same responds as Saleh Alkarim; use 4 C overnight..