Western Blot Protocol for GABA B Receptor 1 Antibody (NB300-145)

Procedure Guide for NB 300-145 Polyclonal Anti-GABA_R1

Western Blot Procedure

1) Resolve ~90 mg of whole brain or cerebral cortex tissue lysates on a 4-20% Tris-Glycine gel.
2) Transfer to nitrocellulose membranes.
3) Block membranes for 1 hour, at room temperature (RT), with TBST (Tween-20 at 0.05%) containing 5% non-fat dry milk (NFDM).
4) Incubate membranes overnight, at 4 degrees C, in NB 300-145 diluted 1:500 in 1X TBS + 1% BSA.
5) Wash with TBST (Tween-20 at 0.1%) wash for 35 minutes at RT (1 X 15 minutes, 2 X 10 minutes).
6) Incubate membranes with HRP-conjugated goat-anti-rabbit IgG, diluted in 1X TBS + 1% BSA, for 30 minutes at RT.
7) Wash with TBST wash for 35 minutes at RT (1 X 15 minutes, 2 X 10 minutes).
8) Using Alpha Innotech ChemiGlow ECL Kit, mix equal volumes of the two reagents. Incubate the membranes in the luminol reageant for ~30-60 seconds.
9) Develop accordingly.