Hoefer PR625 Immunoblot - Manual (Page 18)

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Appendix B:
Detection using ECL Western
blotting detection systems
Hoefer recommends that you perform labeling
and detection on blots following the instructions
provided in your Western blotting labeling and
detection kit. Below is a general protocol based
on the GE Healthcare (formerly Amersham
Biosciences) ECL Western Blotting Detection Kit.
There are several ECL Western blotting detec-
tion systems available.
The following general protocol is suggested:
1
After electrophoresis and transfer of separated
proteins to nitrocellulose or PVDF (low fluorescent
Hybond LFP for highest sensitivity) membrane, block
unspecific sites with a suitable blocking solution.
2
Blocking is followed by two quick washes in
PBS/0.1%Tween (PBS-T).
3
Immerse and incubate the membrane with an antigen-
specific primary antibody of optimized concentration.
4
Remove the membrane with two quick washes of
PBS-T followed by 2 longer washes (2 × 5 min with
rocking).