using ABTS with HRP-conj for immunoscreening of lambda library

[Sebastian W. Bunka]

robert at chironex.jcu.edu.au wrote:
: Hi,

: Just a quick one. Has anyone used ABTS and HRP-conjugated secondary antibodies on Amersham Hybond N+ filters for the purpose of immunoscreening a lambda gt11 
: library ?

: Any pointers would be appreciated.

ABTS is producing a soluble product. For precipitating substrates (Western/
Colony blot) use for ex. chlornaphtol or a mixture of CN and DAB
(Diaminobenzidine) = much more sensitive !!!!.
Stocks:
DAB: 20 mg/ml in DMF (Dimethylformamide)
CN:  300 mg/ml ethanol
store at -20 degC

Wash: 0.9% NaCl; 10 mM Tris pH 8.0
Blocking: Wash + 0.5% gelatine

After! blocking wash w/ Wash + 0.05% Tween 20

Prior developing add 0.1 ml CN to 10 ml 50 mM Tris pH 7.6, filter thru
paper filter (white precipitate! ), add 0.2 ml DAB, add 10 ul 30% H2O2
Add to NC membrane and develop (takes few seconds !!)
Stop by washing in H2O. Photograph (the almost black color is fading
overnight to a brown color).

Cheers, SWB

                      [ Sebastian.Bunka at vu-wien.ac.at ]
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