klenchin at facstaff.REMOVE_TO_REPLY.wisc.edu (Dima Klenchin) wrote:
>our lab is finally moving from I-125 to ECL-based Westerns.
>While I am sure every kit works, I am wondering what you guys
>would recommend to use most. Right now we can think of a reagents from
>Boehringer, Amersham, Pierce and NEN. Of course, other things
>being equal, the cheaper the better.
If you do not require the absolute highest sensitivity you may want to try the
Diaminobenzidine/Nickel method. Peroxidase turns the DAB into a redish
precipitate, which reduces the Ni2+ ions to elementary nickel, giving a black
precipitate directly on the blot. As you can (particularily on PVDF membranes)
incubate for 2 h or more if required, the loss of sensitivity compared to ECL
is not that big, but the method is a lot simpler and cheaper.
Mix 4 ml of Ni buffer (3.03 g Tris-HCl pH 7.6 , 7.3 g NaCl, 0.475 g NiCl2 * 6 H2O
ad 100 ml) with 1 ml 10% DAB (carefull with that stuff), 35 ml water and 2 ul 30%
H2O2 and pour onto your blot. Spots should develope in a few minutes to 2 h,
depending on concentration of your antigen. When you have optimal signal, stop
the reaction by washing the blot with 2-3 changes of distilled water.
There is a Au/Ag amplification step described for this method to increase
sensitivity, but in my hands it does not work very well, it is also not normally
required.
