Bio-Rad Immun-Star™ AP Chemiluminescence Kits User Manual
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2.3 Working Solutions (based on 200 cm
2
of
membrane; one large blot, or four mini gel blots)
This kit has been designed to blot 2,500 cm
2
of membrane, or
approximately 50 mini blots. The Working Solutions section of
this manual has been based on enough of each solution to
blot 200 cm
2
of membrane at 0.5 ml of solution per cm
2
for
each step, except the substrate/enhancer step, which
requires only 0.05 ml per cm
2
. For practical reference,
200 cm
2
of membrane is approximately the size of one large
western blot, or four mini gel western blots. If a larger or
smaller number of blots is used, vary the quantity of reagents
by the appropriate amount.
Since any blotting procedure is best when it is performed with
fresh solutions, it is preferable to make up the solutions
needed for the day’s blots. If solutions containing proteins are
made and held over a period of days, they must be refrigerat-
ed. On the day that the solutions are going to be used, they
should be warmed to room temperature before use to avoid
adding any new variables to the blotting procedure.
Tips
It is important that enough reagent be used to cover the entire
blot. In order to make most efficient use of the reagents, the
container used for wash and incubation steps should fit the
blot as closely as possible.
The enhancer used in this procedure is difficult to wash from
containers, so care should be taken to use disposable or