Hi,
Use Carnation Non-Fat milk powder 5% in your blocking solution. Block O/N
in the cold room or 1 hour at RT and use liberal volume (ie 200 mL). I've
found that with most of the chemiluminesence detection systems that ALL
other blockers are crap. You can by this (Carnation milk powder) in a local
grocery store (in NA). Make SURE it's non-fat.
Andor
""Jayakumar, R"" <R.Jayakumar at RoswellPark.org> wrote in message
news:97101976F8A044468CA74FE11883B90E020489C6 at VISTA.roswellpark.org...
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 performed 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. I am using the same primary and secondary antibodies as I use for
CSPD.
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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