Recombinant Anti-PKA R2/PKR2 antibody [Y116] (ab32514)
Key features and details
- Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
- Rabbit monoclonal [Y116] to PKA R2/PKR2
- Suitable for: Flow Cyt (Intra), IP, WB, IHC-P, ICC/IF
- Knockout validated
- Reacts with: Human
Related conjugates and formulations
Overview
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Product name
Anti-PKA R2/PKR2 antibody [Y116]
See all PKA R2/PKR2 primary antibodies -
Description
Rabbit monoclonal [Y116] to PKA R2/PKR2 -
Host species
Rabbit -
Specificity
The antibody recognises PKA RII. It does not cross-react with other cAMP-dependent kinase. -
Tested applications
Suitable for: Flow Cyt (Intra), IP, WB, IHC-P, ICC/IFmore details -
Species reactivity
Reacts with: Human
Predicted to work with: Cow -
Immunogen
Synthetic peptide within Human PKA R2/PKR2 aa 350-450 (C terminal). The exact sequence is proprietary.
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Positive control
- WB: K562, HeLa and Jurkat cell lysates. IHC-P: Human colon cancer tissue. IP: K562 whole cell lysate. Flow Cyt (intra): MCF7 cells. ICC/IF: HeLa cells.
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General notes
This product is a recombinant monoclonal antibody, which offers several advantages including:
- - High batch-to-batch consistency and reproducibility
- - Improved sensitivity and specificity
- - Long-term security of supply
- - Animal-free production
Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMAb® patents.
Mouse, Rat: We have preliminary internal testing data to indicate this antibody may not react with these species. Please contact us for more information.
Properties
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Form
Liquid -
Storage instructions
Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C long term. Avoid freeze / thaw cycle. -
Storage buffer
pH: 7.20
Preservative: 0.01% Sodium azide
Constituents: 59% PBS, 40% Glycerol (glycerin, glycerine), 0.05% BSA -
Concentration information loading...
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Purity
Protein A purified -
Clonality
Monoclonal -
Clone number
Y116 -
Isotype
IgG -
Research areas
Associated products
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Alternative Versions
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Isotype control
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KO cell lines
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KO cell lysates
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Positive Controls
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Recombinant Protein
Applications
The Abpromise guarantee
Our Abpromise guarantee covers the use of ab32514 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 |
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Flow Cyt (Intra) |
1/20.
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IP |
1/20.
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WB | (1) |
1/10000. Detects a band of approximately 52 kDa (predicted molecular weight: 45 kDa).
For unpurified use at 1/10000 - 1/50000. |
IHC-P |
1/100. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
See IHC antigen retrieval protocols. For unpurified use at 1/100 - 1/250. |
|
ICC/IF |
1/500.
For unpurified use at 1/100 - 1/250. |
Notes |
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Flow Cyt (Intra)
1/20. |
IP
1/20. |
WB
1/10000. Detects a band of approximately 52 kDa (predicted molecular weight: 45 kDa). For unpurified use at 1/10000 - 1/50000. |
IHC-P
1/100. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol. See IHC antigen retrieval protocols. For unpurified use at 1/100 - 1/250. |
ICC/IF
1/500. For unpurified use at 1/100 - 1/250. |
Target
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Function
Regulatory subunit of the cAMP-dependent protein kinases involved in cAMP signaling in cells. Type II regulatory chains mediate membrane association by binding to anchoring proteins, including the MAP2 kinase. -
Tissue specificity
Four types of regulatory chains are found: I-alpha, I-beta, II-alpha, and II-beta. Their expression varies among tissues and is in some cases constitutive and in others inducible. -
Sequence similarities
Belongs to the cAMP-dependent kinase regulatory chain family.
Contains 2 cyclic nucleotide-binding domains. -
Post-translational
modificationsPhosphorylated by the activated catalytic chain. -
Cellular localization
Cytoplasm. Cell membrane. Colocalizes with PJA2 in the cytoplasm and the cell membrane. - Information by UniProt
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Database links
- Entrez Gene: 5576 Human
- Omim: 176910 Human
- SwissProt: P13861 Human
- Unigene: 631923 Human
- Unigene: 731573 Human
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Alternative names
- cAMP dependent protein kinase regulatory subunit alpha 2 antibody
- cAMP dependent protein kinase regulatory subunit RII alpha antibody
- cAMP dependent protein kinase type II alpha regulatory chain antibody
see all
Images
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All lanes : Anti-PKA R2/PKR2 antibody [Y116] (ab32514) at 1/10000 dilution (Purified)
Lane 1 : K-562 (Human chronic myelogenous leukemia lymphoblast) whole cell lysate
Lane 2 : HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate
Lane 3 : Jurkat (Human T cell leukemia T lymphocyte) whole cell lysate
Lysates/proteins at 20 µg per lane.
Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution
Predicted band size: 45 kDa
Observed band size: 50 kDa why is the actual band size different from the predicted? -
Purified ab32514 at 1/20 dilution (0.5µg) immunoprecipitating PKA R2/PKR2 in K-562 whole cell lysate.
Lane 1 (input): K-562 (Human chronic myelogenous leukemia lymphoblast) whole cell lysate 10µg
Lane 2 (+): ab32514 + K-562 whole cell lysate.
Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab32514 in K-562 whole cell lysate.
VeriBlot for IP Detection Reagent (HRP) (ab131366) (1/1000 dilution) was used for Western blotting.
Blocking Buffer and concentration: 5% NFDM/TBST.
Diluting buffer and concentration: 5% NFDM/TBST.
Observed band size: 50 kDa -
Intracellular Flow Cytometry analysis of MCF7 (Human breast adenocarcinoma epithelial cell) cells labeling PKA R2/PKR2 with purified ab32514 at 1/20 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).
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Immunocytochemistry analysis of HeLa (Human cervix adenocarcinoma epithelial cell) cells labeling PKA R2/PKR2 with purified ab32514 at 1/500 dilution (1.9 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.
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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human colon cancer tissue sections labeling PKA R2/PKR2 with purified ab32514 at 1/100 dilution (0.84 µg/mL). Heat mediated antigen retrieval using Bond™ Epitope Retrieval Solution 2 (pH 9.0). Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) was used as the secondary antibody. Negative control: PBS instead of the primary antibody. Hematoxylin was used as a counterstain.
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All lanes : Anti-PKA R2/PKR2 antibody [Y116] (ab32514) at 1/1000 dilution
Lane 1 : Wild-type HeLa cell lysate
Lane 2 : PRKAR2A knockout HeLa cell lysate
Lane 3 : K-562 cell lysate
Lysates/proteins at 20 µg per lane.
Secondary
All lanes : Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) at 1/10000 dilution
Predicted band size: 45 kDa
Observed band size: 50 kDa why is the actual band size different from the predicted?Lanes 1-3: Merged signal (red and green). Green - ab32514 observed at 50 kDa. Red - loading control ab8245 observed at 36 kDa.
Unpurified ab32514 Anti-PKA R2/PKR2 antibody [Y116] was shown to specifically react with PKA R2/PKR2 in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab265748 (knockout cell lysate ab257607) was used. Wild-type and PKA R2/PKR2 knockout samples were subjected to SDS-PAGE. ab32514 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated at room temperature for 2. 5 hours at 1 in 1000 dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.
Protocols
To our knowledge, customised protocols are not required for this product. Please try the standard protocols listed below and let us know how you get on.
Datasheets and documents
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SDS download
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Datasheet download
References (8)
ab32514 has been referenced in 8 publications.
- Jiang Y et al. Type IIB PKA is highly expressed in β cells and controls cell proliferation via regulating Cyclin D1 expression. FEBS J 289:2865-2876 (2022). PubMed: 34839588
- Peng L et al. Prostaglandin E2 Receptor 4 (EP4) Affects Trophoblast Functions via Activating the cAMP-PKA-pCREB Signaling Pathway at the Maternal-Fetal Interface in Unexplained Recurrent Miscarriage. Int J Mol Sci 22:N/A (2021). PubMed: 34502044
- Xie S et al. miR-1307 promotes hepatocarcinogenesis by CALR-OSTC-endoplasmic reticulum protein folding pathway. iScience 24:103271 (2021). PubMed: 34761190
- Hedderich J et al. Norepinephrine Inhibits the Proliferation of Human Bone Marrow-Derived Mesenchymal Stem Cells via ß2-Adrenoceptor-Mediated ERK1/2 and PKA Phosphorylation. Int J Mol Sci 21:N/A (2020). PubMed: 32486305
- Speichert S et al. Role of Norepinephrine in IL-1ß-Induced Chondrocyte Dedifferentiation under Physioxia. Int J Mol Sci 20:N/A (2019). PubMed: 30861996
- El Bagdadi K et al. Norepinephrine Inhibits Synovial Adipose Stem Cell Chondrogenesis via a2a-Adrenoceptor-Mediated ERK1/2 Activation. Int J Mol Sci 20:N/A (2019). PubMed: 31248037
- Hillman P et al. Dissociation between AKAP3 and PKARII promotes AKAP3 degradation in sperm capacitation. PLoS One 8:e68873 (2013). WB ; Cow . PubMed: 23894359
- Gu L et al. Protein kinase A activation confers resistance to trastuzumab in human breast cancer cell lines. Clin Cancer Res 15:7196-206 (2009). IHC-P ; Human . PubMed: 19920112