On June 29, 2017Abdel-Rahman Al-Absi, Aarhus University Hospital wrote:
5
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.
Western blot
Immunostaining
Not Rated
IP
Not Rated
ELISA
Not Rated
Flow cytometry
Not Rated
Luminex
Not Rated
On November 12, 2014Simon Mølgaard, Aarhus University wrote:
1
This was tested os hippocampal lysates from p20 mice.This showed a very unspecific staining with high background. used at 1:1000
Western blot
Immunostaining
Not Rated
IP
Not Rated
ELISA
Not Rated
Flow cytometry
Not Rated
Luminex
Not Rated
On April 12, 2014Simon Mølgaard, Aarhus University wrote:
4
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.
On June 29, 2017 Abdel-Rahman Al-Absi, Aarhus University Hospital wrote:
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.
On December 9, 2016 Simon Mølgaard wrote:
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.
On November 12, 2014 Simon Mølgaard, Aarhus University wrote:
This was tested os hippocampal lysates from p20 mice.This showed a very unspecific staining with high background. used at 1:1000
On April 12, 2014 Simon Mølgaard, Aarhus University wrote:
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.