Validated using a knockout cell lineRecombinantRabMAb

Recombinant Anti-Caldesmon/CDM antibody [E89] (ab32330)

Submit a review Q&A (6)References (23)
Western blot - Anti-Caldesmon/CDM antibody [E89] (ab32330)
  • Western blot - Anti-Caldesmon/CDM antibody [E89] (ab32330)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Caldesmon/CDM antibody [E89] (ab32330)
  • Immunocytochemistry - Anti-Caldesmon/CDM antibody [E89] (ab32330)
  • Immunoprecipitation - Anti-Caldesmon/CDM antibody [E89] (ab32330)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Caldesmon/CDM antibody [E89] (ab32330)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Caldesmon/CDM antibody [E89] (ab32330)
  • Flow Cytometry (Intracellular) - Anti-Caldesmon/CDM antibody [E89] (ab32330)
  • Anti-Caldesmon/CDM antibody [E89] (ab32330)

Key features and details

  • Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
  • Rabbit monoclonal [E89] to Caldesmon/CDM
  • Suitable for: WB, IHC-P, Flow Cyt (Intra), IP, ICC
  • Knockout validated
  • Reacts with: Mouse, Rat, Human

Conjugates logo Related conjugates and formulations

Alexa Fluor® 488 Alexa Fluor® 555 Alexa Fluor® 647 Carrier Free HRP PE

Overview

  • Product name

    Anti-Caldesmon/CDM antibody [E89]
    See all Caldesmon/CDM primary antibodies

  • Description

    Rabbit monoclonal [E89] to Caldesmon/CDM

  • Host species

    Rabbit

  • Tested applications

    Suitable for: WB, IHC-P, Flow Cyt (Intra), IP, ICCmore details

  • Species reactivity

    Reacts with: Mouse, Rat, Human

  • Immunogen

    Synthetic peptide. This information is proprietary to Abcam and/or its suppliers.

  • Positive control

    • WB: HeLa and NIH/3T3 cell lysates; IHC-P: Human breast carcinoma, Human leiomyoma and mouse kidney tissue; ICC: NIH/3T3 cells; IP: NIH/3T3 whole cell lysate; Flow Cyt (intra): NIH/3T3 cells. WB: HeLa, NIH/3T3 whole cell lysates. Rat Liver lysate.

  • 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
    For more information see here.

    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.

Properties

Applications

The Abpromise guarantee

Our Abpromise guarantee covers the use of ab32330 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
WB
1/10000 - 1/20000. Detects a band of approximately 70 kDa (predicted molecular weight: 93 kDa).
IHC-P
1/100. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
Flow Cyt (Intra)
1/20.

ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody.
IP
1/20.
ICC
1/50.

For unpurified use at 1/250 - 1/500.
Notes
WB
1/10000 - 1/20000. Detects a band of approximately 70 kDa (predicted molecular weight: 93 kDa).
IHC-P
1/100. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
Flow Cyt (Intra)
1/20.

ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody.
IP
1/20.
ICC
1/50.

For unpurified use at 1/250 - 1/500.

Target

  • Function

    Actin- and myosin-binding protein implicated in the regulation of actomyosin interactions in smooth muscle and nonmuscle cells (could act as a bridge between myosin and actin filaments). Stimulates actin binding of tropomyosin which increases the stabilization of actin filament structure. In muscle tissues, inhibits the actomyosin ATPase by binding to F-actin. This inhibition is attenuated by calcium-calmodulin and is potentiated by tropomyosin. Interacts with actin, myosin, two molecules of tropomyosin and with calmodulin. Also play an essential role during cellular mitosis and receptor capping.

  • Tissue specificity

    High-molecular-weight caldesmon (isoform 1) is predominantly expressed in smooth muscles, whereas low-molecular-weight caldesmon (isoforms 2, 3, 4 and 5) are widely distributed in non-muscle tissues and cells. Not expressed in skeletal muscle or heart.

  • Sequence similarities

    Belongs to the caldesmon family.

  • Domain

    The N-terminal part seems to be a myosin/calmodulin-binding domain, and the C-terminal a tropomyosin/actin/calmodulin-binding domain. These two domains are separated by a central helical region in the smooth-muscle form.

  • Post-translational
    modifications

    In non-muscle cells, phosphorylation by CDK1 during mitosis causes caldesmon to dissociate from microfilaments. Phosphorylation reduces caldesmon binding to actin, myosin, and calmodulin as well as its inhibition of actomyosin ATPase activity. Phosphorylation also occurs in both quiescent and dividing smooth muscle cells with similar effects on the interaction with actin and calmodulin and on microfilaments reorganization.

  • Cellular localization

    Cytoplasm > cytoskeleton. Cytoplasm > myofibril. On thin filaments in smooth muscle and on stress fibers in fibroblasts (nonmuscle).
  • Information by UniProt

  • Database links

    • Alternative names

      • CAD antibody
      • CALD 1 antibody
      • CALD1 antibody
      • CALD1_HUMAN antibody
      • Caldesmon 1 antibody
      • Caldesmon 1 Isoform 1 antibody
      • Caldesmon 1 Isoform 2 antibody
      • Caldesmon 1 Isoform 3 antibody
      • Caldesmon 1 Isoform 4 antibody
      • Caldesmon 1 Isoform 5 antibody
      • Caldesmon antibody
      • Caldesmon1 antibody
      • CDM antibody
      • H CAD antibody
      • HCAD antibody
      • L CAD antibody
      • LCAD antibody
      • MGC21352 antibody
      • NAG22 antibody
      see all

    Images

    • Western blot - Anti-Caldesmon/CDM antibody [E89] (ab32330)
      Western blot - Anti-Caldesmon/CDM antibody [E89] (ab32330)
      All lanes : Anti-Caldesmon/CDM antibody [E89] (ab32330) at 1/10000 dilution (Purified)

      Lane 1 : HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate
      Lane 2 : NIH/3T3 (Mouse embryonic fibroblast) whole cell lysate
      Lane 3 : Rat liver lysate

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

      Predicted band size: 93 kDa

    • Western blot - Anti-Caldesmon/CDM antibody [E89] (ab32330)
      Western blot - Anti-Caldesmon/CDM antibody [E89] (ab32330)
      All lanes : Anti-Caldesmon/CDM antibody [E89] (ab32330) at 1/1000 dilution

      Lane 1 : Wild-type HeLa cell lysate
      Lane 2 : CALD1 knockout HeLa cell lysate
      Lane 3 : NIH/3T3 cell lysate
      Lane 4 : HEK-293 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: 93 kDa
      Observed band size: 75 kDa why is the actual band size different from the predicted?



      Lanes 1-4: Merged signal (red and green). Green - ab32330 observed at 75 kDa. Red - loading control ab8245 observed at 36 kDa.

      ab32330 Anti-Caldesmon/CDM antibody [E89] was shown to specifically react with Caldesmon/CDM in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab265026 (knockout cell lysate ab257375) was used. Wild-type and Caldesmon/CDM knockout samples were subjected to SDS-PAGE. ab32330 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C 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.

       

    • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Caldesmon/CDM antibody [E89] (ab32330)
      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Caldesmon/CDM antibody [E89] (ab32330)

      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human leiomyoma tissue sections labeling Caldesmon/CDM with purified ab32330 at 1/100 dilution (1.21 µg/mL). Heat mediated antigen retrieval was performed using Heat mediated antigen retrieval using Bond™ Epitope Retrieval Solution 2 (pH 9.0). Tissue was counterstained with Hematoxylin. Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) secondary antibody was used at 1/0 dilution. PBS instead of the primary antibody was used as the negative control.

      The immunostaining was performed on a Leica Biosystems BOND® RX instrument

    • Immunocytochemistry - Anti-Caldesmon/CDM antibody [E89] (ab32330)
      Immunocytochemistry - Anti-Caldesmon/CDM antibody [E89] (ab32330)

      Immunocytochemistry analysis of NIH/3T3 (Mouse embryonic fibroblast) cells labeling Caldesmon/CDM with purified ab32330 at 1/50 dilution (2.4 µg/mL). Cells were fixed in 100% Methanol 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.

    • Immunoprecipitation - Anti-Caldesmon/CDM antibody [E89] (ab32330)
      Immunoprecipitation - Anti-Caldesmon/CDM antibody [E89] (ab32330)

      Purified ab32330 at 1/20 dilution (0.6µg) immunoprecipitating Caldesmon/CDM in NIH/3T3 whole cell lysate.
      Lane 1 (input): NIH/3T3 (Mouse embryonic fibroblast) whole cell lysate.
      Lane 2 (+): ab32330 + NIH/3T3 whole cell lysate.
      Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab32330 in NIH/3T3 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.

    • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Caldesmon/CDM antibody [E89] (ab32330)
      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Caldesmon/CDM antibody [E89] (ab32330)

      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of mouse kidney tissue sections labeling Caldesmon/CDM with purified ab32330 at 1/100 dilution (1.21 µg/mL). Heat mediated antigen retrieval was performed using Heat mediated antigen retrieval using Bond™ Epitope Retrieval Solution 2 (pH 9.0). Tissue was counterstained with Hematoxylin. Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) secondary antibody was used at 1/0 dilution. PBS instead of the primary antibody was used as the negative control.

      The immunostaining was performed on a Leica Biosystems BOND® RX instrument

    • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Caldesmon/CDM antibody [E89] (ab32330)
      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Caldesmon/CDM antibody [E89] (ab32330)

      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of rat stomach tissue sections labeling Caldesmon/CDM with purified ab32330 at 1/100 dilution (1.21 µg/mL). Heat mediated antigen retrieval was performed using Heat mediated antigen retrieval using Bond™ Epitope Retrieval Solution 2 (pH 9.0). Tissue was counterstained with Hematoxylin. Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) secondary antibody was used at 1/0 dilution. PBS instead of the primary antibody was used as the negative control.

      The immunostaining was performed on a Leica Biosystems BOND® RX instrument

    • Flow Cytometry (Intracellular) - Anti-Caldesmon/CDM antibody [E89] (ab32330)
      Flow Cytometry (Intracellular) - Anti-Caldesmon/CDM antibody [E89] (ab32330)

      Intracellular Flow Cytometry analysis of NIH/3T3 (Mouse embryonic fibroblast) cells labelling Caldesmon/CDM with purified ab32330 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). Unlabelled control - Cell without incubation with primary antibody and secondary antibody (Blue).

       

    • Anti-Caldesmon/CDM antibody [E89] (ab32330)
      Anti-Caldesmon/CDM antibody [E89] (ab32330)

    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.

    Click here to view the general protocols

    References (23)

    Publishing research using ab32330? Please let us know so that we can cite the reference in this datasheet.

    ab32330 has been referenced in 23 publications.

    • Lee LL  et al. Cardiac pericytes function as key vasoactive cells to regulate homeostasis and disease. FEBS Open Bio 11:207-225 (2021). PubMed: 33135334
    • Fardoun M  et al. 7-O-methylpunctatin, a Novel Homoisoflavonoid, Inhibits Phenotypic Switch of Human Arteriolar Smooth Muscle Cells. Biomolecules 9:N/A (2019). PubMed: 31717401
    • Nakagaki-Silva EE  et al. Identification of RBPMS as a mammalian smooth muscle master splicing regulator via proximity of its gene with super-enhancers. Elife 8:N/A (2019). PubMed: 31283468
    • Domke LM & Franke WW The cell-cell junctions of mammalian testes: II. The lamellar smooth muscle monolayer cells of the peritubular wall are laterally connected by vertical adherens junctions-a novel architectonic cell-cell junction system. Cell Tissue Res N/A:N/A (2018). PubMed: 30591979
    • Wu X  et al. Subtype-Specific Tumor-Associated Fibroblasts Contribute to the Pathogenesis of Uterine Leiomyoma. Cancer Res 77:6891-6901 (2017). PubMed: 29055020
    View all Publications for this product

    Customer reviews and Q&As

    Show All Reviews Q&A
    Submit a review Submit a question

    1-6 of 6 Abreviews or Q&A

    Question

    Thank you for your kind reply.

    The customer has also requested some specific instructions or a more precise protocol regarding incubation times and recommended buffers for FACS and IF.

    Also, could you please mention a cell type or cell line that might serve as a good negative control for Caldsmon?

    Any cells known not to express Cladesmon might do.

    Thank you for your kind support.

    Regards,

    Read More
    Answer

    I have attached an image of our Flow cytometry results with ab32330.

    We used untreated HeLa cells as our negative control and Anisomycin-treated HeLa cells as our positive control. The primary dilution was 1:20. Both were permeabilized according to our standard protocol which is described below:

    1. Solutions and Reagents
    1.1. 1X PBS

    1.2. Blocking buffer: 0.5% BSA in 1X PBS

    1.3. 2% paraformaldehyde (1% solution - optional for storing samples)

    1.4. 1X FACS permeabilizing solution (BD Biosciences cat. #340973)

    1.5. Fluorescently-conjugated secondary antibody (various forms)



    2. Protocol
    2.1. Collect 1x106 cells/sample.

    2.2. Wash cells once with blocking buffer.

    2.3. Fix cells with 2% paraformaldehyde and incubate at room temperature for 10 min.

    2.4. Wash cells once with blocking buffer.

    2.5. Add 0.5 ml 1X FACS permeabilizing solution and incubate at room temperature for 10 min.

    2.6. Wash cells once with blocking buffer.

    2.7. Incubate cells in blocking buffer for 30 min at room temperature.

    2.8. Add primary antibody at the appropriate dilution and incubate for 30 min at room temperature.

    2.9. Wash twice with blocking buffer and incubate with fluorescently-conjugated secondary antibody for 30 min at room temperature.

    2.10. Wash cells twice with blocking buffer.

    2.11. Re-suspend cells in 1X PBS and analyze on flow cytometry. Samples can be kept in 1% paraformaldehyde at 4 °C overnight.

    Please let me know if you have any additional questions.

    Read More

    Question

    Dear Tech support

    Could you please specify what is the formal shelf life of product ab32330?
    I know it's guaranteed for 6 month from delivery but how long is good for when stored properly?

    Thank you for your kind advise.
    Regards,

    Read More
    Answer

    Thank you for your enquiry regarding ab32330.

    Generally, antibodies can be stored at -20°C for several years if the storage buffer contains cryoprotective agent and the aliquot size is no smaller than 10 µl. Please always check the product datasheet for storage information.

    However, I would like to emphasize that Abcam can only offer a full guarantee called Abpromise within 6 months of purchase.

    https://www.abcam.com/index.html?pageconfig=resource&rid=10795

    If you need any further assistance in the future, please do not hesitate to contact me.

    Read More

    Question

    The antibody is needed for Flow Cytometry in Human or Mouse.
    Thank you for your help!

    Regards,

    Read More
    Answer

    Thank you for your reply and for kindly providing the further information.

    We have one antibody for detection of Caldesmon which has been tested in flow cytometry and mouse, which I think is the one you already found:

    Caldesmon antibody [E89] (ab32330)

    The sequence we used to raise this antibody has 100% homology with all 6 Caldesmon isoforms. Depending on the level of expression in the customer's sample, they may be able to observe all 6.

    Regrettably, this is therefore not specific for the smaller isoforms of the protein in this case and may not be what the customer is looking for.

    If you have any further questions, please do not hesitate to contact me.

    Read More

    Question

    Is antibody ab32330 caldesmon isoform1 specific? could you please specify what is the isoform specificity and if it's not isoform 1, is there an antibody for this protein?

    Read More
    Answer

    Thank you for contacting us.

    Checking the immunogen sequence for this antibody, this antibody is not specific to Caldesmon isoform 1. Based on the sequence, it might crossreact with isoform 1-6 of human Caldesmon protein.



    We have two antibodies which react only with the heavy form/ Isoform 1:

    ab50016 Anti-Caldesmon antibody [hHCD]

    https://www.abcam.com/CaldesmonCDM-antibody-hHCD-ab50016.html (or use the following: https://www.abcam.com/CaldesmonCDM-antibody-hHCD-ab50016.html).

    and ab11013 Anti-Caldesmon antibody [CALD 5]

    https://www.abcam.com/CaldesmonCDM-antibody-CALD-5-ab11013.html (or use the following: https://www.abcam.com/CaldesmonCDM-antibody-CALD-5-ab11013.html).



    I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

    Use our products? Submit an Abreview. Earn rewards!
    https://www.abcam.com/abreviews

    Read More

    Question

    I am wondering if this antibody (ab32330) recognize the high-molecular weight caldesmon? As shown on your website there are different isoforms in different tissue and I'd like to get antibody for isoform 1.

    Read More
    Answer

    Thank you for contacting us. Our source for this antibody confirms that the antibody does recognize a 120-150 kDa protein in smooth muscle samples. They do not, have a blot image, however. Please do not hesitate to contact us if you need any more advice or information.

    Read More

    Question

     Dear sirs,   I have ordered your product no ab 32330, rabbit monoclonal to Caldesmon. In the datasheet it says IgG as an isotype. Is this supposed to be IgG1 or what does it mean?   With kind regards  

    Read More
    Answer

    Thank you for your patience. The isotype of the anti-Caldesmon antibody is simply rabbit IgG. In immunology, no specific isotype has been determined for rabbits. It is only known for human and mouse. I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

    Read More

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