[Histonet] AP binding

[Nancy.Walker <@t> sanofi-synthelabo.com]

Hello,

We're trying a new binding technique using Alkaline Phosphatase fusion
proteins. This technique makes it possible to do binding experiments with
proteins that are unavailble as a radioiodinated form. For initial trials
we are using Neurotensin-AP, for which we have  radioactive binding tests
to verify the AP results.

Protocol: Staining of tissue sections :
1.    Rehydrate the sections for 10 min in 100 µl of HBHA.
2.    Incubate for 2 hours in the AP fusion Neurotensin at 100 nM diluted
in HBS (cover all sections on the slide) at room temperature in a moist
chamber.
3.    Rinse 4 times in cold HBS.
4.    Fix for 15 sec in Acetone-Formaldehyde fixative.
5.    Wash the sections 6 times in HBS.
6.    Incubate the sections in preheated HBS, in a 65°C water bath, for 15
min.
7.    Wash the sections once in AP reaction buffer.
8.    Add BCIP/TNBT substrate to cover the sections on the slide. Incubate
in the dark at room temperature.

Recipe:
·     HBHA (Hepes-Buffered Hanks' Solution) :
         BSA????..0.5 mg/ml
         NaN3????0.1% (w/v)
         Hepes????20 mM
         Adjust pH to 7. Prepare solution in Hanks'.
·     HBS (Hepes Buffered Saline)
         NaCl????.150 mM
         Hepes????20 mM
         Adjust pH to 7. Prepare solution in distilled water.
·     Acetone-Formaldehyde Fixative :
         Acetone?????.?.60% (v/v)
         Formaldehyde????3% (v/v)
         Hepes???????.20 mM
         Adjust pH to 7. Prepare solution in distilled water.
·     AP Reaction Buffer :
         Tris-HCl??????100 mM
         NaCl???????...100 mM
         MgCl2???????.50 mM
         Adjust pH to 9.5. Prepare solution in distilled water.
BCIP/TNBT :
Substrate delivered by CEMICON INTERNATIONAL.

First 2 trials we got nothing.........
Does anyone have a protocol or suggestions??

thanks and have a good weekend,

Nancy Walker
Molecular Biology Scientist

Sanofi-Synthelbo Research
B.P. 37 Labége Innopole
31676 LABEGE CEDEX FRANCE

nancy.walker <@t> sanofi-synthelabo.com
tel : (33)561004179  fax :(33)561004001