hi..
I am having problems with high background on my Immobilon-P PVDF membranes. I use 7-12 % mini SDS-PAGE gels. The PVDF (Immo-P) membranes are wetted and transferred on using Tris+ Glycine+ 20 % methanol transfer buffer, at 90 V for 90 min. I use PBS-T for washing and 1% caesin in PBS-T for blocking (1 hour). Incubations with antibodies were performd at recommended periods of 3 hours (RT) or overnight (4C) for primary and 1 hour for secondary. During development of blot with the ready-to-use chemiluminescent substrate CDP-Star (from Roche), I observe very high background in addition to the bands of interest. Comparitive studies with another substrate CSPD (from Roche), no background is observed at all though the bands of interest is not as sensitive as that obtained from CDP-star.
Has anybody in this group faced similar problems with CDP-star substrate before. If so what remedies were practiced? Any ideas would be useful. My thanks for your anticipated suggestions.
Jai
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