Table 3.

Western blotting protocols for transporters

	NET	VMAT 1	VMAT 2
Total protein	10 μg	40 μg	40 μg
Type of gel	8% Tris-Glycine gel	4-12% NuPAGE Bis-Tris gel	4-12% NuPAGE Bis-Tris gel
Blocking buffer	I-block buffer (1X PBS, 0.1%tween-20, 0.02% sodium azide, 0.2% I-block dry powder (Applied Biosystems, Foster City, CA, USA)	TBS-T and 5% normal donkey serum (Jackson Immuno, Westgrove, PA, USA)	TBS-T and 5% dry milk
Primary antibody	Rabbit anti-human NET polyclonal antibody (kind gift from Dr David Christie, University of Auckland, refs 13, 14), 1:250, 72 h at 4 °C	Goat anti-human VMAT 1 polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA), 1:2000, 1 h at RT	Rabbit anti-human VMAT 2 polyclonal antibody (Chemicon, Temecula, CA, USA)
Secondary antibody	Alkaline phosphatase conjugated anti-rabbit IgG, 1:5000, 45 min at RT	Horseradish peroxidase conjugated anti-goat IgG, 1:2000, 1 h at RT	Horseradish peroxidase conjugated anti-goat IgG, 1:20 000, 1 h at RT
Immunodetection system	CDP-Star chemiluminescent method (Applied Biosystems)	Chemiluminescent method and using NIH image 1.62 software to perform densitometry analyses	Chemiluminescent method and using NIH image 1.62 software to perform densitometry analyses

NET, noradrenaline transporter; RT, room temperature.