Overview

  • Product name
    Anti-CaMKII antibody [EP1829Y]
    See all CaMKII primary antibodies
  • Description
    Rabbit monoclonal [EP1829Y] to CaMKII
  • Host species
    Rabbit
  • Specificity
    The peptide immunogen is highly conserved between CaMKII alpha, beta, gamma, and delta.
  • Tested applications
    Suitable for: ICC/IF, WB, IHC-Pmore details
    Unsuitable for: Flow Cyt or IP
  • Species reactivity
    Reacts with: Mouse, Rat, Human
  • Immunogen

    Synthetic peptide within Human CaMKII aa 200-300. The exact sequence is proprietary.
    (Peptide available as ab199551)

  • Positive control
    • WB: Mouse brain, rat brain and human fetal brain tisse lysates. IHC-P: Human hepatocellulas carcinoma tissue. ICC/IF: U87-MG and PC12 cells.
  • 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 ab52476 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 1/250.
WB 1/1000 - 1/5000. Detects a band of approximately 45, 70 kDa (predicted molecular weight: 54 kDa).

For unpurified use at 1/20000.

IHC-P 1/500.
  • Application notes
    Is unsuitable for Flow Cyt or IP.
  • Target

    Images

    • Anti-CaMKII antibody [EP1829Y] (ab52476) at 1/5000 dilution (purified) + Human fetal brain tissue lysate at 20 µg

      Secondary
      HRP-conjugated anti-rabbit IgG, specific to the non-reduced form of IgG at 1/1000 dilution

      Predicted band size: 54 kDa
      Observed band size: 45,70 kDa
      why is the actual band size different from the predicted?



      Blocking buffer and concentration: 5% NFDM/TBST.

      Diluting buffer and concentration: 5% NFDM /TBST.

    • All lanes : Anti-CaMKII antibody [EP1829Y] (ab52476) at 1/1000 dilution (purified)

      Lane 1 : Mouse brain tissue lysate
      Lane 2 : Rat brain tissue lysate

      Lysates/proteins at 20 µg per lane.

      Secondary
      All lanes : HRP-conjugated anti-rabbit IgG, specific to the non-reduced form of IgG at 1/1000 dilution

      Predicted band size: 54 kDa
      Observed band size: 45,70 kDa why is the actual band size different from the predicted?



      Blocking buffer and concentration: 5% NFDM/TBST.

      Diluting buffer and concentration: 5% NFDM /TBST.

    • Anti-CaMKII antibody [EP1829Y] (ab52476) at 1/20000 dilution (unpurified) + Mouse brain lysate at 10 µg

      Secondary
      Goat anti-rabbit HRP labeled at 1/2000 dilution

      Predicted band size: 54 kDa
      Observed band size: 45,70 kDa why is the actual band size different from the predicted?

    • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human hepatocellular carcinoma tissue labelling CaMKII with purified ab52476 at 1/500. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9. A prediluted HRP-polymer conjugated anti-rabbit IgG was used as the secondary antibody. Negative control using PBS instead of primary antibody. Counterstained with hematoxylin.

    • Immunocytochemistry/Immunofluorescence analysis of U87-MG cells labelling CaMKII with purified ab52476 at 1/250. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% aTriton X-100. An Alexa Fluor® 555-conjugated goat anti-rabbit IgG (1/500) was used as the secondary antibody. DAPI (blue) was used as the nuclear counterstain.

      Control: primary antibody (1/250) and secondary antibody, ab150113, an Alexa Fluor® 488-conjugated goat anti-mouse IgG (1/500).

    • ICC/IF image of unpurified ab52476 stained PC12 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 (ab52476, 1µg/ml) overnight at +4°C. The secondary antibody (green)ÿwas 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:
    • Xiao YF  et al. FKBP12.6 protects heart from AngII-induced hypertrophy through inhibiting Ca2+ /calmodulin-mediated signalling pathways in vivo and in vitro. J Cell Mol Med 22:3638-3651 (2018). Read more (PubMed: 29682889) »
    • Zahra A  et al. Memantine rescues prenatal citalopram exposure-induced striatal and social abnormalities in mice. Exp Neurol 307:145-154 (2018). Read more (PubMed: 29913137) »
    See all 33 Publications for this product

    Customer reviews and Q&As

    1-7 of 7 Abreviews or Q&A

    Application
    Western blot
    Sample
    Rat Tissue lysate - whole (Rat Artery (Mesenteric/Cerebral/Renal))
    Gel Running Conditions
    Reduced Denaturing (10% gel)
    Loading amount
    30 µg
    Specification
    Rat Artery (Mesenteric/Cerebral/Renal)
    Blocking step
    Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 22°C

    Mr. Edward Humphries

    Verified customer

    Submitted May 24 2017

    Answer

    Thanks so much for your reply.
    How is the tissue fixed and prepared before staining? If it is PFA fixed for more than ˜15 minutes, I'd suggest trying an antigen retrieval treatment in pH6 sodium citrate buffer, and a 1:100 dilution of primary antibody. As we have only tested formalin-fixed paraffin-embedded tissue with ab52476, it may not work with other types of tissue unfortunately.
    If the tissue you're using is perfused with PFA for fixation (we label this as "IHC-FoFr"), we have the CaMKII beta antibody ab34703 which will be suitable-
    https://www.abcam.com/camkii-beta-antibody-ab34703.html
    For fresh frozen sections (IHC-Fr), I'd suggest ab22609, however it is a mouse monoclonal so there may be some background due to the anti-mouse secondary antibody-
    https://www.abcam.com/camkii-antibody-6g9-ab22609.html
    If the mouse-on-mouse staining with ab22609 does cause background, you can reduce this with an anti-mouse F(ab) fragment block like ab6668, or by conjugating a fluorophore directly to the primary antibody. We have conjugation kits like ab102884 which can be used for this purpose.
    I hope that this information will be useful, but if you have further questions or need anything else, please let me know and I'll be happy to help.

    Read More

    Answer

    Thank you very much for contacting us and letting us know about the trouble with this antibody.
    I do have a few questions, so that I can better understand the situation.
    1) How is the mouse brain tissue fixed and processed? Ab52476 has only been tested on formalin-fixed and paraffin-embedded tissue, so we are not sure whether it works with cryosections or perfused-fixed tissue.
    2) Do you perform any antigen retrieval steps?
    3) What kind of blocking solution do you use?
    4) We've tested the antibody on paraffin-embedded tissue up to 1:100 dilution (chromogenic detection though). Have you tried higher a higher concentration than 1:200?
    5) I've looked through our records and believe I've found your order- could you confirm the order or PO# that this antibody was received on?
    We haven't received any other recent reports of this antibody not working, so I'm not sure that the batch is bad. Even though the protocol has worked well with other primaries in the past, there may be some optimization required in order to see results with this antibody, so once I have the above details I may have some suggestions.
    I look forward to hearing from you. Please let me know if you have any further questions or if there is anything else that we can do for you, and I'll be happy to help.

    Read More

    Answer

    Thank you for contacting us.
    I am sorry to hear that the antibody is giving problems.

    As we discussed over the phone,I would suggest adding protease inhibitors to the lysis buffer in order to prevent the lysed proteins to be digested and degraded by proteases.

    Please let me know if this helps to improve your results. If not, please let me know also your order or PO number, so that we can discuss other options of resolving the issue.

    I wish you good luck and look forward to hear back from you.

    Read More

    Answer

    Thank you for your enquiry. I can confirm that ab52476 CaMKII antibody [EP1829Y] recognizes all subunits of the protein, since the peptide immunogen is highly conserved between CaMKII-alpha, -beta, -gamma, and -omega. I hope this will be helpful to you. If you have any further questions, please do not hesitate to contact us.

    Read More
    Abcam guarantees this product to work in the species/application used in this Abreview.
    Application
    Western blot
    Sample
    Human Cell lysate - whole cell (huh-7)
    Loading amount
    15000 cells
    Specification
    huh-7
    Treatment
    cells expressing recombitant viral protein
    Gel Running Conditions
    Reduced Denaturing
    Blocking step
    Milk as blocking agent for 1 minute(s) · Concentration: 0.5% · Temperature: 20°C

    Abcam user community

    Verified customer

    Submitted Aug 04 2009

    Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"

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