Recombinant
RabMAb

Recombinant Anti-cAMP Protein Kinase Catalytic subunit antibody [EP2102Y] (ab76238)

Overview

  • Product name

    Anti-cAMP Protein Kinase Catalytic subunit antibody [EP2102Y]
    See all cAMP Protein Kinase Catalytic subunit primary antibodies
  • Description

    Rabbit monoclonal [EP2102Y] to cAMP Protein Kinase Catalytic subunit
  • Host species

    Rabbit
  • Tested applications

    Suitable for: ICC/IF, WB, IP, IHC-P, Flow Cytmore details
  • Species reactivity

    Reacts with: Mouse, Rat, Human
  • Immunogen

    Synthetic peptide within Human cAMP Protein Kinase Catalytic subunit aa 300-400 (C terminal). The exact sequence is proprietary.

  • Positive control

    • ICC/IF: HeLa cells FC: MCF-7 and HeLa cells IHC-P: Rat stomach tissue, Mouse cerebrum tissue, Human testis and thyroid carcinoma tissue WB: MCF-7, HeLa, NIH/3T3 and C6 cell lysates
  • General notes

     

     

    Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMab® patents.

    We are constantly working hard to ensure we provide our customers with best in class antibodies. As a result of this work we are pleased to now offer this antibody in purified format. We are in the process of updating our datasheets. The purified format is designated 'PUR' on our product labels. If you have any questions regarding this update, please contact our Scientific Support team.

    This product is a recombinant rabbit monoclonal antibody.

Properties

Applications

Our Abpromise guarantee covers the use of ab76238 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Abreviews Notes
ICC/IF 1/100 - 1/250.
WB 1/20000 - 1/200000. Detects a band of approximately 42 kDa (predicted molecular weight: 46 kDa).
IP 1/40 - 1/50.
IHC-P 1/750. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

See IHC antigen retrieval protocols.

For unpurified use at 1/100 - 1/250.

Use of HRP-conjugated or polymerized HRP secondary antibodies recommended, stronger signals have been found using the polymerized HRP secondary.

Flow Cyt 1/60 - 1/80.

ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody.

Target

  • Function

    Phosphorylates 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 specificity

    Isoform 1 is ubiquitous. Isoform 2 is sperm-specific and is enriched in pachytene spermatocytes but is not detected in round spermatids.
  • Sequence similarities

    Belongs 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
    modifications

    Asn-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 localization

    Cytoplasm. 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.
  • Information by UniProt
  • Database links

  • Alternative names

    • cAMP dependent protein kinase alpha catalytic subunit antibody
    • cAMP dependent protein kinase beta catalytic subunit antibody
    • cAMP dependent protein kinase catalytic beta subunit isoform 4ab antibody
    • cAMP dependent protein kinase catalytic subunit alpha antibody
    • cAMP dependent protein kinase catalytic subunit alpha, isoform 1 antibody
    • cAMP dependent protein kinase catalytic subunit beta antibody
    • cAMP-dependent protein kinase catalytic subunit alpha antibody
    • KAPCA_HUMAN antibody
    • PKA C alpha antibody
    • PKA C beta antibody
    • PKA C-alpha antibody
    • PKACA antibody
    • PKACB antibody
    • PPNAD4 antibody
    • PRKACA antibody
    • PRKACAA antibody
    • PRKACB antibody
    • Protein kinase A catalytic subunit alpha antibody
    • Protein kinase A catalytic subunit antibody
    • Protein kinase A catalytic subunit beta antibody
    • Protein kinase, cAMP dependent, catalytic, alpha antibody
    • Protein kinase, cAMP dependent, catalytic, beta antibody
    see all

Images

  • All lanes : Anti-cAMP Protein Kinase Catalytic subunit antibody [EP2102Y] (ab76238) at 1/20000 dilution (Purified)

    Lane 1 : MCF7 (Human breast adenocarcinoma epithelial cell) whole cell lysates
    Lane 2 : NIH/3T3 (Mouse embryonic fibroblast) whole cell lysates
    Lane 3 : C6 (Rat glial tumor glial cell) whole cell lysates

    Lysates/proteins at 15 µg per lane.

    Secondary
    All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

    Predicted band size: 46 kDa
    Observed band size: 42 kDa
    why is the actual band size different from the predicted?

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of rat stomach tissue sections labeling cAMP Protein Kinase Catalytic subunit with purified ab76238 at 1/750 dilution. Heat mediated antigen retrieval was performed Perform heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0). ImmunoHistoProbe one step HRP Polymer (ready to use) was used as the secondary antibody. Negative control: PBS instead of the primary antibody. Hematoxylin was used as a counterstain.
  • ab76238 (purified) at 1/40 dilution (2 µg) immunoprecipitating cAMP Protein Kinase Catalytic subunit in MCF-7 whole cell lysate.
    Lane 1 (input): MCF-7 (Human breast adenocarcinoma epithelial cell) whole cell lysate 10 µg
    Lane 2 (+): ab76238 & MCF-7 whole cell lysate
    Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab76238 in MCF-7 whole cell lysate
    For western blotting, VeriBlot for IP Detection Reagent (HRP) (ab131366) was used at 1/1000 dilution.
    Blocking and diluting buffer: 5% NFDM/TBST.
  • Flow Cytometry analysis of MCF-7 (Human breast adenocarcinoma epithelial cell) cells labeling cAMP Protein Kinase Catalytic subunit with purified ab76238 at 1/80 dilution (10 µg/ml) (Red). Cells were fixed with 4% Paraformaldehyde and permeabilised with 90% Methanol. A Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) secondary antibody was used at 1/2000. Isotype control - Rabbit monoclonal IgG (Black). Unlabeled control - Cell without incubation with primary antibody and secondary antibody (Blue).
  • Immunocytochemistry/ Immunofluorescence analysis of HeLa (Human cervix adenocarcinoma epithelial cell) cells labeling cAMP Protein Kinase Catalytic subunit with purified ab76238 at 1:100 dilution (8.0 µg/ml). Cells were fixed in 4% Paraformaldehyde and permeabilized with 0.1% tritonX-100. Cells were counterstained with Ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) 1:200 (2.5 µg/ml). Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) was used as the secondary antibody at 1:1000 (2 µg/ml) dilution. DAPI (blue) was used as nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of mouse cerebrum tissue sections labeling cAMP Protein Kinase Catalytic subunit with purified ab76238 at 1/750 dilution. Heat mediated antigen retrieval was performed Perform heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0). ImmunoHistoProbe one step HRP Polymer (ready to use) was used as the secondary antibody. Negative control: PBS instead of the primary antibody. Hematoxylin was used as a counterstain.
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human thyroid carcinoma tissue sections labeling cAMP Protein Kinase Catalytic subunit with purified ab76238 at 1/750 dilution. Heat mediated antigen retrieval was performed Perform heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0). ImmunoHistoProbe one step HRP Polymer (ready to use) was used as the secondary antibody. Negative control: PBS instead of the primary antibody. Hematoxylin was used as a counterstain.
  • All lanes : Anti-cAMP Protein Kinase Catalytic subunit antibody [EP2102Y] (ab76238) at 1/200000 dilution (unpurified)

    Lane 1 : HeLa cell lysate
    Lane 2 : MCF-7 cell lysate

    Lysates/proteins at 10 µg per lane.

    Secondary
    All lanes : goat anti-rabbit HRP at 1/1000 dilution

    Predicted band size: 46 kDa
    Observed band size: 42 kDa why is the actual band size different from the predicted?

  • ab76238 (unpurified), at a 1/100 dilution, staining human cAMP Protein Kinase Catalytic Subunit in testis by Immunohistochemistry, Formalin/PFA-fixed paraffin-embedded tissue.

    Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

  • ICC/IF image of ab76238 (unpurified) 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.

  • Overlay histogram showing HeLa cells stained with ab76238 (unpurified) (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

This product has been referenced in:

  • Yu YC  et al. Resveratrol Improves Brain-Gut Axis by Regulation of 5-HT-Dependent Signaling in the Rat Model of Irritable Bowel Syndrome. Front Cell Neurosci 13:30 (2019). Read more (PubMed: 30800058) »
  • Yan G  et al. A RIPK3-PGE2 Circuit Mediates Myeloid-Derived Suppressor Cell-Potentiated Colorectal Carcinogenesis. Cancer Res 78:5586-5599 (2018). Read more (PubMed: 30012671) »
See all 8 Publications for this product

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