I'm trying to detect total S-nitrosylation in SH-SY5Y cell culture using Sodium Nitroprusside (200, 400uM). I've treated cells with SNP for 15 min and isolated proteins using lysis buffer containing 150mM NaCl, 100mM Hepes (ph=8), 1mM EDTA, 0.1mM neocuprione, 1% NP-40, protease inhibitor cocktail, 20mM NEM, 0.01% SDS. Free thiols were blocked using 20mM MMTS dissolved in buffer containing Hepes-NaOH (pH 7.7), EDTA and neocuprione at 55 degrees for 45 min. Excess of blocking buffer was removed using acetone precipitation. Nitrosylated proteins were reduced using 50 mM ascorbate and 10 uM copper sulfate at room temperature for 20 min. Reduced thiols were tagged with 4mM biotin-HDPD at room temperature for 1 h. Using immunoblot method, I'm not able to see the difference between control and SNP intensities. Signal intensity for control is still high. When I performed treatment of SNP with already lysed proteins for 20 min at room temperature, I'm able to see the significant difference in signal intensities. Please suggest me how can I see significant difference in control and SNP treated cells and not in cell lysates. 

Thanks...