The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Use a concentration of 5 µg/ml.
Use at an assay dependent concentration. PubMed: 25086961
1/500 - 1/1000. Detects a band of approximately 46 kDa (predicted molecular weight: 46 kDa).
Use at an assay dependent concentration. PubMed: 20603614
Use a concentration of 5 µg/ml. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
The arrestins are a family of proteins that are important for regulating signal transduction within cells. Arrestins are part of a conserved two step mechanism for regulating the activity of G-protein coupled receptors (GPCRs). In response to a stimulus, GPCRs activate a heterotrimeric G protein. In order to turn off this response, or adapt to a constant stimulus, activated receptors need to be silenced. The first step is phosphorylation by a class of serine/threonine kinases called G protein coupled receptor kinases (GRKs). This phosphorylation specifically marks the activated receptor for arrestin binding. Once arrestin is bound to the receptor it is unable to signal further. Recent research continues to expand the known actions of arrestins, which can bind to other classes of receptors and can directly activate signaling pathways on their own.
Different arrestins (visual arrestin (or Arrestin 1), beta-arrestin 1 (or Arrestin 2) and beta-arrestin 2 (or Arrestin 3) can reduce the activity of their target GPCRs in several different ways.
Cytoplasm. Note: Associated with plasma membrane, as well as with endodomes and lysosomes during endocytosis.
IHC image of ab64817 staining in normal human hippocampus formalin fixed paraffin embedded tissue section, performed on a Leica BondTM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab64817, 5µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.
For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.
ICC/IF image of ab64817 stained PC12 cells. The cells were 4% formaldehyde fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab64817, 5µg/ml) overnight at +4°C. The secondary antibody (green) was ab96899, DyLight® 488 goat anti-rabbit IgG (H+L) used at a 1/250 dilution for 1h.Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.
Tian X et al. The a-Arrestin ARRDC3 Regulates the Endosomal Residence Time and Intracellular Signaling of the ß2-Adrenergic Receptor. J Biol Chem291:14510-25 (2016).
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