Overview

  • Product name
    Anti-PKC beta 1 + PKC beta 2 (phospho T500) antibody
    See all PKC beta 1 + PKC beta 2 primary antibodies
  • Description
    Rabbit polyclonal to PKC beta 1 + PKC beta 2 (phospho T500)
  • Host species
    Rabbit
  • Specificity
    This antibody cross-reacts with PKC alpha [pT497] (88% homologous) and partially cross-reacts with PKC gamma [pT514] (63% homologous) and epsilon [pT566] (75% homologous), as determined by peptide competition experiments.
  • Tested applications
    Suitable for: ICC/IF, WBmore details
  • Species reactivity
    Reacts with: Rat, Human
    Predicted to work with: Mouse
  • Immunogen

    Synthetic phosphopeptide derived from a region of human PKC beta 1 & 2 that contains threonine 500.

  • Positive control
    • K562 cells treated with PMA, a phorbol ester.
  • General notes


    Protein Kinase C beta (PKC beta) is an 80 kDa member of the conventional group (cPKCs: sensitive to diacylglycerol, phosphotidylserine and phorbol esters) of the PKC family of serine/threonine kinases that are involved in a wide range of physiological processes including mitogenesis, cell survival, transcriptional regulation and tumor promotion. PKC beta has been implicated in diabetes and carcinogenesis. PKC beta isoforms 1 & 2 are phosphorylated on three sites, threonine 500 in the activation loop, beta 1 threonine 642 (beta 2 641) in the turn loop and beta 1 serine 661 (beta 2 660) in the hydrophobic loop. Phosphorylation of PKC beta 1 & 2 on threonine 500 by PDK1 is a prerequisite for its autophosphoylation and catalytic competence.

Properties

  • Form
    Liquid
  • Storage instructions
    Shipped at 4°C. Upon delivery aliquot and store at -20°C or -80°C. Avoid repeated freeze / thaw cycles.
  • Storage buffer
    pH: 7.30
    Preservative: 0.05% Sodium azide
    Constituents: PBS, 50% Glycerol, 0.1% BSA
  • Concentration information loading...
  • Purity
    Immunogen affinity purified
  • Purification notes
    The antibody has been negatively preadsorbed using a non-phosphopeptide corresponding to the site of phosphorylation to remove antibody that is reactive with non-phosphorylated PKC beta. The final product is generated by affinity chromatography using a PKC beta-derived peptide that is phosphorylated at threonine 500.
  • Primary antibody notes
    Protein Kinase C beta (PKC beta) is an 80 kDa member of the conventional group (cPKCs: sensitive to diacylglycerol, phosphotidylserine and phorbol esters) of the PKC family of serine/threonine kinases that are involved in a wide range of physiological processes including mitogenesis, cell survival, transcriptional regulation and tumor promotion. PKC beta has been implicated in diabetes and carcinogenesis. PKC beta isoforms 1 & 2 are phosphorylated on three sites, threonine 500 in the activation loop, beta 1 threonine 642 (beta 2 641) in the turn loop and beta 1 serine 661 (beta 2 660) in the hydrophobic loop. Phosphorylation of PKC beta 1 & 2 on threonine 500 by PDK1 is a prerequisite for its autophosphoylation and catalytic competence.
  • Clonality
    Polyclonal
  • Isotype
    IgG
  • Research areas

Applications

Our Abpromise guarantee covers the use of ab5817 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 1/1000.

Punctate staining observed.

WB 1/1000. Detects a band of approximately 80 kDa.

Target

  • Function
    Calcium-activated and phospholipid-dependent serine/threonine-protein kinase involved in various processes such as regulation of the B-cell receptor (BCR) signalosome, apoptosis and transcription regulation. Plays a key role in B-cell activation and function by regulating BCR-induced NF-kappa-B activation and B-cell suvival. Required for recruitment and activation of the IKK kinase to lipid rafts and mediates phosphorylation of CARD11/CARMA1 at 'Ser-559', 'Ser-644' and 'Ser-652', leading to activate the NF-kappa-B signaling. Involved in apoptosis following oxidative damage: in case of oxidative conditions, specifically phosphorylates 'Ser-36' of isoform p66Shc of SHC1, leading to mitochondrial accumulation of p66Shc, where p66Shc acts as a reactive oxygen species producer. Acts as a coactivator of androgen receptor (ANDR)-dependent transcription, by being recruited to ANDR target genes and specifically mediating phosphorylation of 'Thr-6' of histone H3 (H3T6ph), a specific tag for epigenetic transcriptional activation that prevents demethylation of histone H3 'Lys-4' (H3K4me) by LSD1/KDM1A. Also involved in triglyceride homeostasis. Serves as the receptor for phorbol esters, a class of tumor promoters.
  • 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.
  • Post-translational
    modifications
    Phosphorylation on Thr-500 within the activation loop renders it competent to autophosphorylate. Subsequent autophosphorylation of Thr-642 maintains catalytic competence, and autophosphorylation on Ser-661 appears to release the kinase into the cytosol. Autophosphorylation on other sites i.e. in the N-terminal and hinge regions have no effect on enzyme activity.
  • Cellular localization
    Cytoplasm. Nucleus. Membrane.
  • Information by UniProt
  • Database links
  • Alternative names
    • KPCB_HUMAN antibody
    • PKC beta antibody
    • PKC-B antibody
    • PKC-beta antibody
    • PKCB antibody
    • Prkcb antibody
    • PRKCB I antibody
    • PRKCB II antibody
    • PRKCB1 antibody
    • PRKCB2 antibody
    • Protein kinase C beta 1 antibody
    • Protein kinase C beta 2 antibody
    • Protein kinase C beta antibody
    • Protein kinase C beta type antibody
    • protein kinase C, beta 1 polypeptide antibody
    see all

Images

  • Peptide Competition and Phosphatase Treatment: Lysates prepared from K562 cells stimulated with PMA were resolved by SDS-PAGE on a 10% polyacrylamide gel and transferred to PVDF. Membranes were either left untreated (1-4) or treated with lambda phosphatase (5), blocked with a 5% BSA-TBST buffer for one hour at room temperature, and incubated with ab5817 antibody for two hours at room temperature in a 3% BSA-TBST buffer, following prior incubation with: no peptide (1, 5), the non-phosphopeptide corresponding to the immunogen (2), a generic phosphothreonine-containing peptide (3), or, the phosphopeptide immunogen (4). After washing, membranes were incubated with goat F(ab’ 2 anti-rabbit IgG HRP conjugate and bands were detected using the Pierce SuperSignalTM method. The data show that only the peptide corresponding to PKC beta 1 & 2  [pT500] blocks the antibody signal. The data also show that phosphatase stripping eliminates the signal, verifying that the antibody is phospho-specific.

    Peptide Competition and Phosphatase Treatment: Lysates prepared from K562 cells stimulated with PMA were resolved by SDS-PAGE on a 10% polyacrylamide gel and transferred to PVDF. Membranes were either left untreated (1-4) or treated with lambda phosphatase (5), blocked with a 5% BSA-TBST buffer for one hour at room temperature, and incubated with ab5817 antibody for two hours at room temperature in a 3% BSA-TBST buffer, following prior incubation with: no peptide (1, 5), the non-phosphopeptide corresponding to the immunogen (2), a generic phosphothreonine-containing peptide (3), or, the phosphopeptide immunogen (4). After washing, membranes were incubated with goat F(ab’ 2 anti-rabbit IgG HRP conjugate and bands were detected using the Pierce SuperSignalTM method. The data show that only the peptide corresponding to PKC beta 1 & 2 [pT500] blocks the antibody signal. The data also show that phosphatase stripping eliminates the signal, verifying that the antibody is phospho-specific.

References

This product has been referenced in:
  • Molè D  et al. Protein kinase C: a putative new target for the control of human medullary thyroid carcinoma cell proliferation in vitro. Endocrinology 153:2088-98 (2012). WB ; Human . Read more (PubMed: 22374978) »
  • Spalding AC  et al. Inhibition of protein kinase Cbeta by enzastaurin enhances radiation cytotoxicity in pancreatic cancer. Clin Cancer Res 13:6827-33 (2007). Read more (PubMed: 18006785) »
See all 2 Publications for this product

Customer reviews and Q&As

1-8 of 8 Abreviews or Q&A

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunocytochemistry/ Immunofluorescence
Sample
Rat Cell (Cerebellar neurons in culture)
Specification
Cerebellar neurons in culture
Fixative
Formaldehyde
Blocking step
Gelatin from fish as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 0.25

Abcam user community

Verified customer

Submitted Apr 09 2007

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
Human Cell lysate - whole cell (BxPC3 cells)
Loading amount
20 µg
Specification
BxPC3 cells
Treatment
10% FBS
Blocking step
BSA as blocking agent for 16 hour(s) and 0 minute(s) · Concentration: 5%

Abcam user community

Verified customer

Submitted Oct 23 2006

Answer

Further to a follow up email from our source of this antibody I have ascertained that A431 cells are apparently NOT a good choice for the detection of PKC beta. According to them the cells have been shown to express PKC alpha, delta and zeta but not PKC beta. They question the specificity of the antibody that you have been using to detect PKC beta; perhaps it is detecting generic/pan PKC? They suggest that many of the antibodies on the market demonstrate this reactivity. Jurkat or K562 cells are the best positive control cell lines according to their recommendation. There are likely to be many other cell lines in the literature. However, A431 cells are NOT suitable according to their recommendations. Additional info: K562 and Jurkats were treated with PMA, 100 nM for 10 min.; K562 had the higher expression level and better looking signal so were used those for validation purposes. In the absence of any stimulation, one should be able to detect the phosphorylation of PKCbI&II (in response to endogenous stimuli, e.g., cAMP and others). I recommend that he use Jurkat cells treated with PMA as positive control. Let me know if I can assist further. In light of this information I would like to recommend that an alternative positive control is employed. I hope this information helps, please do not hesitate to contact us if you need any more advice or information.

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Answer

Thank you for getting back to me. I have been unable to obtain a satisfactory answer from the source of this antibody. However, I see no reason why the positive control that you have been using would not be suitable. Furthermore the approach that you are using is one I that I would recommend. I am certainly prepared to offer you a credit note against this purchase provided that the antibody was purchased within the past 90 days. If this is the case please email me details of the order including the order number and date of purchase. I look forward to hearing from you.

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Question

BATCH NUMBER 172133 ORDER NUMBER PO104494 DESCRIPTION OF THE PROBLEM I am not getting any signal at the expected size around 80kD. The only thing that comes up in a very long exposure is a weak band at around 66kD. This is not PKC beta as confirmed by blotting with control PKC beta antibody. SAMPLE Mouse B cell extract PRIMARY ANTIBODY used 1:500 in blocking buffer over night at 4 degrees wash in TBS-T DETECTION METHOD ECL POSITIVE AND NEGATIVE CONTROLS USED that is one of the things I would like to know from you ANTIBODY STORAGE CONDITIONS I stored it at -20 upon arrival, however, it was at room temperature when it arrived, because it was not shipped on dry ice. SAMPLE PREPARATION cells lyzed in buffer containing 150mM NaCl, 50mM Tris, 1mM EDTA, 10% glycerol, 2%ODG (detergent) SDS-sample-buffer containing 2-Mercaptoethanol added and sample boiled prior to gel-loading AMOUNT OF PROTEIN LOADED 30ug ELECTROPHORESIS/GEL CONDITIONS SDS-PAGE 9% TRANSFER AND BLOCKING CONDITIONS Tris-glycine buffer, semi-dry blotting 2h, transfer confirmed by Ponceau-stain block in 5%milk in TBS-T SECONDARY ANTIBODY anti-rabbit-HRP 1h RT(worked well on other membrane developed in parallel) HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 1 HAVE YOU RUN A "NO PRIMARY" CONTROL? No DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes ADDITIONAL NOTES Could you please tell the specific conditions how you have used the antibody (how much protein on gel, what dilution, buffer etc). Also, is there something that I can use as apositive control to make sure that the antibody actually works? I am a bit concerned, because it arrived at room temperature. Thank you, Alina

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Answer

Thank you for your enquiry. I am sorry to hear that you have been having difficulties with this antibody. I have read your technical questionaire and I have a few comments. The approach that you are employing is largely one that I would recommend. I am however concerned that you are not detecting the phosphorylated form of PKC beta given as a result of the low level of phosphorylation of this protein in B cells. We recommend that PMA stimulated K562 cells are used to guarantee high levels of phosphorylation and good signal by western blotting. Unfortunately we do not sell this as a pre-prepared lysate. However, I would like to suggest that a stimulated cell line is incorporated into your experiments to guarantee that you have phosphorylated PKC on your blot for targeting by the antigen. I would also like to recommend that you incorporate protease inhibitors in your lysate preparation as the lack of signal may be the result of protein degradation. The western blotting conditions employed using this antibody are detailed on the antibody datasheet: Membranes were blocked with 5% BSA-TBST and incubated with the antibody diluted in 3% BSA/TBST. Should you continue to experience difficulties please get back in touch with me.

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Answer

Thank you for your enquiry. This testing has not been performed as far as I know, but with some research I have found the following information: PKC alpha shows ~79% homology within the range of the immunogen sequence. PKC beta I shows 50% homology within the range of the immunogen sequence. PKC delta shows ~18% homology within the range of the immunogen sequence. PKC epsilon shows 50% homology within the range of the immunogen sequence. PKC gamma shows ~64% homology within the range of the immunogen sequence. I hope this information helps, please do not hesitate to contact us if you need any more advice or information.

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Answer

Thank you for your enquiry. Are you interested in detecting PKC beta 1 or 2? Ab5785 detects PKC beta 2 phosphorylated at T641, where as ab5817 detects PKC beta 1 and 2 phosphorylated at T500. For both of these antibodies there are Western blot images on the online datasheets, and we guarantee that the antibodies will work in Western blotting with human samples. I hope this helps. If you have any additional questions, please contact us again.

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Answer

Thank you for your enquiry. Useful working concentration range of PMA is 1–100 nM. You may need to consider testing the half-maximal effective dose of PMA in your experimental system. Please take a look at the following website, you may find some useful information on PMA and effective concentration: http://www.promega.com/cnotes/cn001/cn001_10.pdf

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