The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
1/50000 - 1/200000. Detects a band of approximately 42 kDa (predicted molecular weight: 46 kDa).
1/100 - 1/250. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.
Use of HRP-conjugated or polymerized HRP secondary antibodies recommended, stronger signals have been found using the polymerized HRP secondary.
1/100 - 1/250.
ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody.
FunctionPhosphorylates a large number of substrates in the cytoplasm and the nucleus. Regulates the abundance of compartmentalized pools of its regulatory subunits through phosphorylation of PJA2 which binds and ubiquitinates these subunits, leading to their subsequent proteolysis. Phosphorylates CDC25B, ABL1, NFKB1, CLDN3, PSMC5/RPT6, PJA2, RYR2, RORA and VASP. RORA is activated by phosphorylation. Required for glucose-mediated adipogenic differentiation increase and osteogenic differentiation inhibition from osteoblasts. Involved in the regulation of platelets in response to thrombin and collagen; maintains circulating platelets in a resting state by phosphorylating proteins in numerous platelet inhibitory pathways when in complex with NF-kappa-B (NFKB1 and NFKB2) and I-kappa-B-alpha (NFKBIA), but thrombin and collagen disrupt these complexes and free active PRKACA stimulates platelets and leads to platelet aggregation by phosphorylating VASP. Prevents the antiproliferative and anti-invasive effects of alpha-difluoromethylornithine in breast cancer cells when activated. RYR2 channel activity is potentiated by phosphorylation in presence of luminal Ca(2+), leading to reduced amplitude and increased frequency of store overload-induced Ca(2+) release (SOICR) characterized by an increased rate of Ca(2+) release and propagation velocity of spontaneous Ca(2+) waves, despite reduced wave amplitude and resting cytosolic Ca(2+). PSMC5/RPT6 activation by phosphorylation stimulates proteasome. Negatively regulates tight junctions (TJs) in ovarian cancer cells via CLDN3 phosphorylation. NFKB1 phosphorylation promotes NF-kappa-B p50-p50 DNA binding. Involved in embryonic development by down-regulating the Hedgehog (Hh) signaling pathway that determines embryo pattern formation and morphogenesis. Prevents meiosis resumption in prophase-arrested oocytes via CDC25B inactivation by phosphorylation. May also regulate rapid eye movement (REM) sleep in the pedunculopontine tegmental (PPT). Phosphorylates APOBEC3G and AICDA. Isoform 2 phosphorylates and activates ABL1 in sperm flagellum to promote spermatozoa capacitation.
Tissue specificityIsoform 1 is ubiquitous. Isoform 2 is sperm-specific and is enriched in pachytene spermatocytes but is not detected in round spermatids.
Sequence similaritiesBelongs to the protein kinase superfamily. AGC Ser/Thr protein kinase family. cAMP subfamily. Contains 1 AGC-kinase C-terminal domain. Contains 1 protein kinase domain.
Post-translational modificationsAsn-3 is partially deaminated to Asp giving rise to 2 major isoelectric variants, called CB and CA respectively. Autophosphorylated. Phosphorylation is enhanced by vitamin K(2). Phosphorylated on threonine and serine residues. Phosphorylation on Thr-198 is required for full activity. Phosphorylated at Tyr-331 by activated receptor tyrosine kinases EGFR and PDGFR; this increases catalytic efficienncy.
Cellular localizationCytoplasm. Cell membrane. Nucleus. Mitochondrion. Translocates into the nucleus (monomeric catalytic subunit). The inactive holoenzyme is found in the cytoplasm. Distributed throughout the cytoplasm in meiotically incompetent oocytes. Associated to mitochondrion as meiotic competence is acquired. Aggregates around the germinal vesicles (GV) at the immature GV stage oocytes and Cell projection, cilium, flagellum. Expressed in the midpiece region of the sperm flagellum.
ICC/IF image of ab76238 stained HeLa cells. The cells were 4% formaldehyde (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 (ab76238, neat) 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.
Flow Cytometry-Anti-cAMP Protein Kinase Catalytic subunit antibody [EP2102Y](ab76238)
Overlay histogram showing HeLa cells stained with ab76238 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab76238, 1/100 dilution) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-rabbit IgG (H+L) (ab96899) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was rabbit IgG (monoclonal) (1µg/1x106 cells) used under the same conditions. Unlabelled sample (blue line). Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter. This antibody gave a positive signal in HeLa cells fixed with 4% paraformaldehyde (10 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.
References for Anti-cAMP Protein Kinase Catalytic subunit antibody [EP2102Y] (ab76238)
This product has been referenced in:
Ouyang C et al. Transforming Growth Factor (TGF)-ß-activated Kinase 1 (TAK1) Activation Requires Phosphorylation of Serine 412 by Protein Kinase A Catalytic Subunit a (PKACa) and X-linked Protein Kinase (PRKX). J Biol Chem289:24226-37 (2014).
Read more (PubMed: 25028512) »
Geuss LR et al. Paramagnetic beads and magnetically mediated strain enhance cardiomyogenesis in mouse embryoid bodies. PLoS One9:e113982 (2014).
Read more (PubMed: 25501004) »