The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Use at an assay dependent concentration.
Use a concentration of 1 - 5 µg/ml. Detects a band of approximately 34 kDa (predicted molecular weight: 34 kDa).
Use a concentration of 5 µg/ml. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
Functionp35 is a neuron specific activator of CDK5. The complex p35/CDK5 is required for neurite outgrowth and cortical lamination. Activator of TPKII.
Tissue specificityBrain and neuron specific.
Sequence similaritiesBelongs to the cyclin-dependent kinase 5 activator family.
Post-translational modificationsThe p35 form is proteolytically cleaved by calpain, giving rise to the p25 form. P35 has a 5 to 10 fold shorter half-life compared to p25. The conversion results in deregulation of the CDK5 kinase: p25/CDK5 kinase displays an increased and altered tau phosphorylation in comparison to the p35/CDK5 kinase in vivo. Probably myristoylated. The Gly-2-Ala mutant is absent of the cell periphery, suggesting that a proper myristoylation signal is essential for the proper distribution of p35.
Cellular localizationCell membrane. In the primary cortical neurons, p35 is present in the peripheries and nerve terminals and Nucleus. Cytoplasm > perinuclear region. The conversion of p35 to p25 relocalizes the protein from the cell periphery to the cytoplasm, in nuclear and perinuclear regions. In the primary cortical neurons, p25 is primarily concentrated in the cell soma and is largely absent from neurites.
IHC image of ab66064 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 ab66064, 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.
References for Anti-p35 antibody (ab66064)
This product has been referenced in:
Xie W et al. Phosphorylation of kinase insert domain receptor by cyclin-dependent kinase 5 at serine 229 is associated with invasive behavior and poor prognosis in prolactin pituitary adenomas. Oncotarget7:50883-50894 (2016).
Read more (PubMed: 27438154) »
Hou C et al. Effects of an intravitreal injection of interleukin-35-expressing plasmid on pro-inflammatory and anti-inflammatory cytokines. Int J Mol Med38:713-20 (2016).
Read more (PubMed: 27460435) »