ab16653 is raised against an immunizing peptide that recognises mGluR5 (phospho S996). This serine residue is numbered as 996 rather than as 1016 (which is how it is numbered on the database for rat mGluR5; Swiss Prot ID P31424), as the first 20aa of the protein are part of a cleaved signal peptide sequence.
ab16653 specifically detects mGluR5 (phospho S996) protein expression in mouse hippocampal lysate at the expected band size (~130kDa). However, another stronger band is observed at >200kDa with ab16653 as well as with a positive control Rabbit polyclonal mGluR5 antibody. We are unsure of the identity of this larger band. However, both the 130kDa and 200kDa bands are blocked by preincubation with the immunizing peptide for ab16653.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Use a concentration of 1 - 3 µg/ml. Detects a band of approximately 130 kDa (predicted molecular weight: 130 kDa).
Use a concentration of 1 µg/ml.
G-protein coupled receptor for glutamate. Ligand binding causes a conformation change that triggers signaling via guanine nucleotide-binding proteins (G proteins) and modulates the activity of down-stream effectors. Signaling activates a phosphatidylinositol-calcium second messenger system and generates a calcium-activated chloride current. Plays an important role in the regulation of synaptic plasticity and the modulation of the neural network activity.
Belongs to the G-protein coupled receptor 3 family.
ICC/IF image of ab16653 stained rat PC12 cells. The cells were methanol fixed (5 min), permabilised in PBS-T (20 min) and incubated with the antibody (ab16653, 1µg/ml) for 1h at room temperature. 1%BSA / 10% normal goat serum / 0.3M glycine was used to block non-specific protein-protein interactions. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red). DAPI was used to stain the cell nuclei (blue).
Immunohistochemistry (PFA perfusion fixed frozen sections) - Anti-Metabotropic Glutamate Receptor 5 (phospho S996) antibody (ab16653)This image is courtesy of an abreview submitted by Sophie Pezet, Laboratoire de Neurobiologie, France
IHC-FoFr image of Metaobotropic Glutamate Receptor 5 (pS996) staining on rat brain sections using ab16653 (1:300). The sections used came from animals perfused fixed with Paraformaldehyde 4% with 15% of a solution of saturated picric acid, in phosphate buffer 0.1M. Following postfixation in the same fixative overnight, the brains were cryoprotected in sucrose 30% overnight. Brains were then cut using a cryostat and the immunostainings were performed using the ‘free floating’ technique.