Overview

  • Product name
  • Description
    Rabbit polyclonal to PKC
  • Host species
    Rabbit
  • Specificity
    ab19031 recognizes the alpha, beta and gamma isoforms of PKC.
  • Tested applications
    Suitable for: ICC/IF, WBmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Human
  • Immunogen

    Synthetic peptide corresponding to Mouse PKC (C terminal). Synthetic peptide corresponding to the C-terminus of mouse PKC ß1

Properties

Applications

Our Abpromise guarantee covers the use of ab19031 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 Use a concentration of 1 - 5 µg/ml.
WB Use a concentration of 1 - 4 µg/ml. Predicted molecular weight: 82 kDa.

Target

  • Function
    Calcium-activated, phospholipid- and diacylglycerol (DAG)-dependent serine/threonine-protein kinase that is involved in positive and negative regulation of cell proliferation, apoptosis, differentiation, migration and adhesion, tumorigenesis, cardiac hypertrophy, angiogenesis, platelet function and inflammation, by directly phosphorylating targets such as RAF1, BCL2, CSPG4, TNNT2/CTNT, or activating signaling cascade involving MAPK1/3 (ERK1/2) and RAP1GAP. Involved in cell proliferation and cell growth arrest by positive and negative regulation of the cell cycle. Can promote cell growth by phosphorylating and activating RAF1, which mediates the activation of the MAPK/ERK signaling cascade, and/or by up-regulating CDKN1A, which facilitates active cyclin-dependent kinase (CDK) complex formation in glioma cells. In intestinal cells stimulated by the phorbol ester PMA, can trigger a cell cycle arrest program which is associated with the accumulation of the hyper-phosphorylated growth-suppressive form of RB1 and induction of the CDK inhibitors CDKN1A and CDKN1B. Exhibits anti-apoptotic function in glioma cells and protects them from apoptosis by suppressing the p53/TP53-mediated activation of IGFBP3, and in leukemia cells mediates anti-apoptotic action by phosphorylating BCL2. During macrophage differentiation induced by macrophage colony-stimulating factor (CSF1), is translocated to the nucleus and is associated with macrophage development. After wounding, translocates from focal contacts to lamellipodia and participates in the modulation of desmosomal adhesion. Plays a role in cell motility by phosphorylating CSPG4, which induces association of CSPG4 with extensive lamellipodia at the cell periphery and polarization of the cell accompanied by increases in cell motility. Is highly expressed in a number of cancer cells where it can act as a tumor promoter and is implicated in malignant phenotypes of several tumors such as gliomas and breast cancers. Negatively regulates myocardial contractility and positively regulates angiogenesis, platelet aggregation and thrombus formation in arteries. Mediates hypertrophic growth of neonatal cardiomyocytes, in part through a MAPK1/3 (ERK1/2)-dependent signaling pathway, and upon PMA treatment, is required to induce cardiomyocyte hypertrophy up to heart failure and death, by increasing protein synthesis, protein-DNA ratio and cell surface area. Regulates cardiomyocyte function by phosphorylating cardiac troponin T (TNNT2/CTNT), which induces significant reduction in actomyosin ATPase activity, myofilament calcium sensitivity and myocardial contractility. In angiogenesis, is required for full endothelial cell migration, adhesion to vitronectin (VTN), and vascular endothelial growth factor A (VEGFA)-dependent regulation of kinase activation and vascular tube formation. Involved in the stabilization of VEGFA mRNA at post-transcriptional level and mediates VEGFA-induced cell proliferation. In the regulation of calcium-induced platelet aggregation, mediates signals from the CD36/GP4 receptor for granule release, and activates the integrin heterodimer ITGA2B-ITGB3 through the RAP1GAP pathway for adhesion. During response to lipopolysaccharides (LPS), may regulate selective LPS-induced macrophage functions involved in host defense and inflammation. But in some inflammatory responses, may negatively regulate NF-kappa-B-induced genes, through IL1A-dependent induction of NF-kappa-B inhibitor alpha (NFKBIA/IKBA). Upon stimulation with 12-O-tetradecanoylphorbol-13-acetate (TPA), phosphorylates EIF4G1, which modulates EIF4G1 binding to MKNK1 and may be involved in the regulation of EIF4E phosphorylation. Phosphorylates KIT, leading to inhibition of KIT activity. Phosphorylates ATF2 which promotes cooperation between ATF2 and JUN, activating transcription.
  • Sequence similarities
    Belongs to the protein kinase superfamily. AGC Ser/Thr protein kinase family. PKC subfamily.
    Contains 1 AGC-kinase C-terminal domain.
    Contains 1 C2 domain.
    Contains 2 phorbol-ester/DAG-type zinc fingers.
    Contains 1 protein kinase domain.
  • Cellular localization
    Cytoplasm. Cell membrane. Mitochondrion membrane. Nucleus.
  • Information by UniProt
  • Database links
  • Alternative names
    • KPCA_HUMAN antibody
    • PKC alpha antibody
    • PKC beta antibody
    • PKC delta antibody
    • PKC epsilon antibody
    • PKC gamma antibody
    • PKC zeta antibody
    • PKC-A antibody
    • PKC-alpha antibody
    • PKC2 antibody
    • PKCA antibody
    • PKCB antibody
    • PKCD antibody
    • PKCE antibody
    • PKCG antibody
    • PRKCA antibody
    • PRKCB antibody
    • PRKCD antibody
    • PRKCE antibody
    • PRKCG antibody
    • PRKCZ antibody
    • Protein kinase C alpha antibody
    • Protein kinase C alpha type antibody
    • Protein kinase C beta antibody
    • Protein kinase C delta antibody
    • Protein kinase C epsilon antibody
    • Protein kinase C gamma antibody
    • Protein kinase C zeta antibody
    see all

Images

  • Anti-PKC antibody (ab19031) at 0.5 µg/ml + Cell lysate prepared from rat kidney cells

    Predicted band size: 82 kDa

  • ICC/IF image of ab19031 stained MCF7 cells. The cells were 4% PFA 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 (ab19031, 1µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 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.

References

This product has been referenced in:
  • Yu L  et al. Extracorporeal Shock Wave Rebuilt Subchondral Bone In Vivo and Activated Wnt5a/Ca2+Signaling In Vitro. Biomed Res Int 2017:1404650 (2017). Read more (PubMed: 29164146) »
  • Rivera-Gonzalez GC  et al. Skin Adipocyte Stem Cell Self-Renewal Is Regulated by a PDGFA/AKT-Signaling Axis. Cell Stem Cell 19:738-751 (2016). Read more (PubMed: 27746098) »
See all 4 Publications for this product

Customer reviews and Q&As

Answer

Thank you for your enquiry.

I can confirm that any publications that we are aware of will be listed on the datasheet.I'm sorry there are none to provide on this occasion for ab19031. We have a dedicated team who spend time reviewing the literature to find articles that have used our products. However, in many cases, regrettably we do not have access to all the articles mentioning our products, or subscriptions to all the journals that publish them.

I am pleased to provide the protocol usedto test the antibody inwestern blotting. However, please note this may require some individual optimization by the end user.

Solutions and Reagents:

Transfer Buffer:
25 mM Tris-base (pH 8.5), 0.2 M Glycin, 20% methanol

Cell Extract Buffer:
50 mM Pipes/NaOH (pH 6.5), 2 mM EDTA, 0.1% Chaps, 5 mM DTT, 20 g/ml Leupeptin, 10 ug/ml Pepstatin, 10 ug/ml aprotinin, and 1 mM PMSF.

SDS-PAGE Loading Buffer
62.5 mM Tris-HCl, (pH 6.8), 2% w/v SDS, 10% glycerol, 50 mM DTT, 0.01% w/v bromphenol blue

10X TBS (Tris-Buffered Saline):
To prepare 1 liter of 10X TBS: 24.2 g Tris-base, 80 g NaCl, adjust pH to 7.6 with HCl (use at 1X).

TBS/T Washing Buffer:
1X TBS, 0.1% Tween-20

Blocking Buffer:
1X TBS/T with 5% w/v nonfat dry milk. For 150 ml, add 7.5 g nonfat dry milk into 150 ml TBS/T Washing Buffer.

Primary Antibody Dilution Buffer:
1X TBS/T with 5% nonfat milk. For 20 ml, add 2 ml 10X TBS to 18 ml water, then add 1 g nonfat dry milk and mix well.

Western Blot Detection:
Protein marker, secondary anti-rabbit or anti-mouse antibody conjugated to HRP, chemiluminescent reagent, peroxide.

Protein Blotting:

1.Treat cells by adding fresh media containing regulator for desired time.

2.Aspirate media from cultures, wash cells with 1X PBS, aspirate. Scrape cells into PBS and spin down to pellet.

3.Lyse cells by adding Cell Extract Buffer (one volume of cell pellet, or 100 ul per well of 6-well plate or 500 ul per plate of 10 cm2 plate). Freeze and thaw 3 times. Centrifuge lysate at microcentrifuge using top speed. (˜14000 rpm). Keep the supernatant and discard the pelleted cell debris.

4.Add SDS Loading Buffer and heat to 95-100oC for 5 minutes, cool on ice.

5.Microcentrifuge for 5 minutes.

6.Load 5-20 ul onto SDS-PAGE gel (10 cm x 10 cm).

Note: We recommend loading prestained molecular weight markers to verify electrotransfer.

7.Electrotransfer to nitrocellulose membrane.

Membrane Blocking: Antibody Incubations:
Note: Volumes are for 10 cm x 10 cm of membrane. For different sized membranes, adjust volumes accordingly

1.Incubate membrane in 25 ml of Blocking Buffer for 1 hour at room temperature.
2.Wash 3 times for 5 min each with 15 ml of TBS/T.
3.Incubate membrane and primary antibody (at the appropriate dilution) in 10 ml Primary Antibody Dilution Buffer with gentle agitation overnight at 4oC.
4. Wash 3 times for 5 minutes each with 15 ml of TBS/T.
5. Incubate membrane with HRP-conjugated secondary antibody in 10 ml of Blocking Buffer with gentle agitation for 1 hour at room temperature.
6. Wash membrane as in step 4.
7.Proceed with detection.

Detection of Proteins:
1. Remove the wash buffer and place the blot in a plastic bag or clean tray containing chemiluminescent working solution (0.125 ml/cm2) and peroxide (ECL detection method).
2. Rotate the bag or tray to allow the solution to cover the surface of the membrane for 1-5 minutes.
3. Remove blot from bag or tray and place it between two pieces of write-on acetate transparency film. Smooth over covered blot to remove air bubbles and excess substrate.

Expose to X-ray film. An initial exposure of 10-60 seconds is recommended for film.



Should you have any further questions, please do not hesitate to contact us.

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