GABA A receptor alpha 3, Abcam, ab72446

GABA A RECEPTOR ALPHA 3, RABBIT POLYCLONAL, ABCAM AB72446.
I have used this antibody to do western blot on mouse frozen tissue. I have incubated with this antibody (1:1000) at 4 °C over night, then with secondary antibody at room temperature for 1 hour. The antibody gives nice band and almost no background.

This antibody was recently re-tested in hippocampal lysates from 5 days old mice. Here, a nice signal was observed and no background. I highly recommend using this antibody with the following protocol:

Hippocampus was dissected and placed on ice and the tissue was hereafter lysed in TNE-buffer (0.6 g Tris-Base (10 nM); 4,37 g NaCl (150 nm); 1mL 0,5 M EDTA (1 mM); 5 mL NP40 (1%); ddH2O until 500 mL, pH = 8) using a pellet pestle and centrifuged at 10.000 g for 10 min at 4°C. Samples were kept on ice at all times and protein concentration was determined using the Bicinchoninic acid kit (Sigma-Aldrich, #BCA-1)
Protein samples were separated on a 4-12% Bis-Tris SDS-PAGE (Life technologies, #NPO321BOX) in running buffer (Life Technologies, #NP0002) and electroblotted for 2 hrs onto polyvinylidenedifluoride (PVDF) filters (GE Healthcare). Membranes were subsequently blocked in 0.05 M Tris-base, 0.5 M NaCl, 2% skimmed milk powder and 2% Tween-20 for one hour. After blocking, membranes were washed in washing buffer (10 mM HEPES, 140 mM NaCl, 2 mM CaCl2, 1 mM MgCl2, 0.2% skimmed milk powder, 0.05% Tween-20, pH 7.8) and incubated over night at 4°C with primary antibody diluted in blocking buffer at a concentration of 1:1000.
The next day, following three times ten minutes washing, the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies at RT for 1 hour. Following a final washing step, blots were visualized.

This was tested os hippocampal lysates from p20 mice.This showed a very unspecific staining with high background. used at 1:1000

This antibody works very well for detecting GABAa receptor subtype alpha 3 on tissue. I tested 3 different concentrations: 1:50 1:100 and 1:250. 1:100 is absolutely the best choice.