"Jayakumar, R" wrote:
> 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.
PVDF-membranes should be wetted with pure methanol, washed with
distilled water and then equillibrated with transfer buffer. Gels to
should be equilibrated with transfer buffer for 10-15 min.
Better transfer results than with Towbin buffer are often observed with
Dunn's buffer (10 mM NaHCO3, 3 mM Na2CO3), which is also cheaper.
Another important factor is that tank blotters often give better results
than semi-dry systems. Voltage should be high enough to provide
efficient transfer, but not so high as to cause excessive heating. 30-40
V over 90-120 min works fine for me, even with large membrane proteins.
I assume that PBS-T means PBS with 0.05% Tween 20, which should be ok.
Is the casein purified, or in the form of low fat milk powder? The
latter would be better, as pure proteins often fail to cover all
unspecific sites on the membrane. If milk powder also fails, try fish
skin gelatine which despite its ugly looks often solves desperate cases.
Chemoluminescent assays can result in high backgrounds because of their
high sensitivity. Try reducing the primary and/or secondary antibody
concentration and/or the exposure time of the film. This may well be the
reason why a substrate with lower sensitivity gives cleaner results. In
the end you will have to find a compromise between sensitivity and
background noise. Optimal transfer conditions are a key aspect here.
