In “Commercially available antibodies directed against α-adrenergic receptor subtypes and other G protein-coupled receptors with acceptable selectivity in flow cytometry experiments”, Tripathi et al study the specificity of 8 different antibodies against α-adrenergic receptor (AR) subtypes. The specificity of the antibodies was tested by flow cytometry using human vascular smooth muscle cell. Here, a fluorescent signal was measured when treated with siRNA specific for the α-adrenergic receptor (Human ADRA1A (148) (E-005419-00-0050)) and compared with the fluorescent signal from cells treated with non-targeting RNA.
When using this antibody, a 70% reduction in fluorescence was observed.
This antibody was included in a test of ten commercially available antibodies against alpha-1-adrenergic receptor subtypes. All antibodies were tested using Western blotting of brain and heart tissue from WT mice and knock out controls with genetic deletion of one, two or all three alpha-1-adrenergic receptor subtypes.
None of the antibodies detected a band of the appropriate size, or a band of larger or smaller size, that was present in WT mouse heart or brain and absent in KO heart or brain.
The specificity of this antibody was evaluated in immunohistochemistry of murine ureteres and stomach tissue, as well as western blotting of lysed CHO cells.
Immunohistochemisry showed highly distinct staining patterns in rat ureter and stomach.
However, western blotting of cells stably expressing the α1A receptor in CHO cells showed no difference in signal compared to lysates of cells with no endogenous expression. Thus we conclude that this antibody has no specificity for the target antigen.
Dilutions:
WB: 1:400
IHC: 1:50
Ref: Pradidarcheep et al 2009. Lack of specificity of commercially available antisera
against muscarinergic and adrenergic receptors
On October 3, 2018 Simon Molgaard wrote:
In “Commercially available antibodies directed against α-adrenergic receptor subtypes and other G protein-coupled receptors with acceptable selectivity in flow cytometry experiments”, Tripathi et al study the specificity of 8 different antibodies against α-adrenergic receptor (AR) subtypes. The specificity of the antibodies was tested by flow cytometry using human vascular smooth muscle cell. Here, a fluorescent signal was measured when treated with siRNA specific for the α-adrenergic receptor (Human ADRA1A (148) (E-005419-00-0050)) and compared with the fluorescent signal from cells treated with non-targeting RNA.
When using this antibody, a 70% reduction in fluorescence was observed.
See full description in the article:
https://www.ncbi.nlm.nih.gov/pubmed/26660071
On April 15, 2018 Paul Simpson, UCSF wrote:
This antibody was included in a test of ten commercially available antibodies against alpha-1-adrenergic receptor subtypes. All antibodies were tested using Western blotting of brain and heart tissue from WT mice and knock out controls with genetic deletion of one, two or all three alpha-1-adrenergic receptor subtypes.
None of the antibodies detected a band of the appropriate size, or a band of larger or smaller size, that was present in WT mouse heart or brain and absent in KO heart or brain.
For further information see BC Jensen et al., 2009 (https://link.springer.com/article/10.1007%2Fs00210-008-0368-6)
On February 27, 2018 Wouter H. Lamers wrote:
The specificity of this antibody was evaluated in immunohistochemistry of murine ureteres and stomach tissue, as well as western blotting of lysed CHO cells.
Immunohistochemisry showed highly distinct staining patterns in rat ureter and stomach.
However, western blotting of cells stably expressing the α1A receptor in CHO cells showed no difference in signal compared to lysates of cells with no endogenous expression. Thus we conclude that this antibody has no specificity for the target antigen.
Dilutions:
WB: 1:400
IHC: 1:50
Ref: Pradidarcheep et al 2009. Lack of specificity of commercially available antisera
against muscarinergic and adrenergic receptors
https://www.ncbi.nlm.nih.gov/pubmed/?term=19198807