Overview

  • Product name
    Anti-Calmodulin 1/2/3 antibody [2D1]
    See all Calmodulin 1/2/3 primary antibodies
  • Description
    Mouse monoclonal [2D1] to Calmodulin 1/2/3
  • Host species
    Mouse
  • Specificity
    This antibody detects calmodulin. It does not detect parvalbumin, tropinin, S-100, or myosin light chain kinase (MLCK).By Western blot, this antibody detects a 17 kDa protein representing calmodulin from Dictyostelium cell lysate. Immunohistochemical staining of calmodulin in Dictyostelium cells with this antibody results in staining of the contractile vacuoles.
  • Tested applications
    Suitable for: Flow Cyt, ELISA, WB, IHC-P, ICC, IP, ICC/IFmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Chicken, Cow, Human, Dictyostelium discoideum
    Predicted to work with: Wheat
  • Immunogen

    Other Immunogen Type corresponding to Calmodulin 1/2/3. Calmodulin purified from Dictyostelium discoideum.

  • Positive control
    • WB: Purified Dictyostelium calmodulin IHC: rat brain
  • General notes

     

     

Properties

Applications

Our Abpromise guarantee covers the use of ab2860 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
Flow Cyt Use 2µg for 106 cells.

ab170190 - Mouse monoclonal IgG1, is suitable for use as an isotype control with this antibody.

 

ELISA Use at an assay dependent concentration.
WB 1/500. Detects a band of approximately 17 kDa (predicted molecular weight: 17 kDa).
IHC-P 1/20.
ICC 1/50.
IP Use at an assay dependent concentration.
ICC/IF 1/20.

Target

  • Relevance
    Function: Calmodulin mediates the control of a large number of enzymes and other proteins by Ca(2+). Among the enzymes to be stimulated by the calmodulin-Ca(2+) complex are a number of protein kinases and phosphatases. Together with CEP110 and centrin, is involved in a genetic pathway that regulates the centrosome cycle and progression through cytokinesis.
  • Cellular localization
    Cytoplasm > cytoskeleton > spindle. Cytoplasm > cytoskeleton > spindle pole. Distributed throughout the cell during interphase, but during mitosis becomes dramatically localized to the spindle poles and the spindle microtubules.
  • Database links
  • Form
    There are three genes which encode an identical calcium binding protein which is one of the four subunits of phosphorylase kinase.
  • Alternative names
    • CALM 1 antibody
    • CALM 2 antibody
    • CALM 3 antibody
    • CALM antibody
    • CALM1 antibody
    • CALM2 antibody
    • CALM3 antibody
    • CALML2 antibody
    • calmodulin 1 (phosphorylase kinase, delta) antibody
    • Calmodulin 1 antibody
    • Calmodulin 2 (phosphorylase kinase, delta) antibody
    • Calmodulin 2 antibody
    • Calmodulin 3 (phosphorylase kinase, delta) antibody
    • Calmodulin 3 antibody
    • CAM 2 antibody
    • CAM 3 antibody
    • CAM I antibody
    • CAM1 antibody
    • CAM2 antibody
    • CAM3 antibody
    • CAMB antibody
    • CAMC antibody
    • CAMI antibody
    • CAMII antibody
    • CAMIII antibody
    • CPVT4 antibody
    • DD132 antibody
    • FLJ99410 antibody
    • LP7057 protein antibody
    • PHKD antibody
    • PHKD2 antibody
    • PHKD3 antibody
    • phosphorylase kinase delta antibody
    • phosphorylase kinase, delta subunit antibody
    see all

Images

  • Flow cytometry analysis of Calmodulin showing positive staining in the cytoplasm of PC12 cells compared to an isotype control (blue). Cells were harvested and adjusted to a concentration of 1-5x10^6 cells/ml. Cells were then fixed with 2% paraformaldehyde and washed with PBS. Cells were penetrated by dropping the supernatant and adding 90% methanol followed by incubation for 10 minutes at room temperature. Cells were blocked with a 2% solution of BSA-PBS for 30 min at room temperature and incubated with ab2860 at 2 ug/test for 60 min at room temperature. Cells were then incubated for 40 min at room temperature in the dark using a Dylight 488-conjugated goat anti-mouse IgG (H+L) secondary antibody and re-suspended in PBS for FACS analysis.
  • Flow cytometry analysis of Calmodulin showing positive staining in the cytoplasm of MCF-7 cells compared to an isotype control (blue). Cells were harvested and adjusted to a concentration of 1-5x10^6 cells/ml. Cells were then fixed with 2% paraformaldehyde and washed with PBS. Cells were penetrated by dropping the supernatant and adding 90% methanol followed by incubation for 10 minutes at room temperature. Cells were blocked with a 2% solution of BSA-PBS for 30 min at room temperature and incubated with ab2860 at a dilution of 2 ug/test for 60 min at room temperature. Cells were then incubated for 40 min at room temperature in the dark using a Dylight 488-conjugated goat anti-mouse IgG (H+L) secondary antibody and re-suspended in PBS for FACS analysis.
  • Flow cytometry analysis of Calmodulin showing positive staining in the cytoplasm of C6 cells compared to an isotype control (blue). Cells were harvested and adjusted to a concentration of 1-5x10^6 cells/ml. Cells were then fixed with 2% paraformaldehyde and washed with PBS. Cells were penetrated by dropping the supernatant and adding 90% methanol followed by incubation for 10 minutes at room temperature. Cells were blocked with a 2% solution of BSA-PBS for 30 min at room temperature and incubated with ab2860 at 2 ug/test for 60 min at room temperature. Cells were then incubated for 40 min at room temperature in the dark using a Dylight 488-conjugated goat anti-mouse IgG (H+L) secondary antibody and re-suspended in PBS for FACS analysis.
  • Immunolocalization of calmodulin in rat brain using ab2860 (1:100)
  • Anti-Calmodulin 1/2/3 antibody [2D1] (ab2860) at 1/500 dilution + Purified Dictyostelium calmodulin

    Predicted band size: 17 kDa

  • Overlay histogram showing HeLa cells stained with ab2860 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab2860, 2µg/1x106 cells) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (ab91353, 2µg/1x106 cells ) used under the same conditions. Acquisition of >5,000 events was performed.

  • Immunofluorescent analysis of Calmodulin using Calmodulin Monoclonal antibody (2D1) ab2860 shows staining in HeLa cells. Calmodulin staining (green) F-Actin staining with Phalloidin (red) and nuclei with DAPI (blue) is shown. Cells were grown on chamber slides and fixed with formaldehyde prior to staining. Cells were probed without (control) or with or an antibody recognizing Calmodulin ab2860 at a dilution of 1:20 over night at 4 ?C washed with PBS and incubated with a DyLight-488 conjugated secondary antibody. Images were taken at 60X magnification.

  • Immunofluorescent analysis of Calmodulin using Calmodulin Monoclonal antibody (2D1) ab2860 shows staining in A2058 melanoma cells. Calmodulin staining (green) F-Actin staining with Phalloidin (red) and nuclei with DAPI (blue) is shown. Cells were grown on chamber slides and fixed with formaldehyde prior to staining. Cells were probed without (control) or with or an antibody recognizing Calmodulin ab2860 at a dilution of 1:20 over night at 4 ?C washed with PBS and incubated with a DyLight-488 conjugated secondary antibody. Images were taken at 60X magnification.

  • Immunofluorescent analysis of Calmodulin using Calmodulin Monoclonal antibody (2D1) ab2860 shows staining in C6 glioma cells. Calmodulin staining (green) F-Actin staining with Phalloidin (red) and nuclei with DAPI (blue) is shown. Cells were grown on chamber slides and fixed with formaldehyde prior to staining. Cells were probed without (control) or with or an antibody recognizing Calmodulin ab2860 at a dilution of 1:20 over night at 4 ?C washed with PBS and incubated with a DyLight-488 conjugated secondary antibody. Images were taken at 60X magnification.

  • Immunohistochemistry was performed on normal biopsies of deparaffinized Rat testis tissue. To expose target proteins heat induced antigen retrieval was performed using 10mM sodium citrate (pH6.0) buffer microwaved for 8-15 minutes. Following antigen retrieval tissues were blocked in 3% BSA-PBS for 30 minutes at room temperature. Tissues were then probed at a dilution of 1:20 with a mouse monoclonal antibody recognizing Calmodulin ab2860 or without primary antibody (negative control) overnight at 4°C in a humidified chamber. Tissues were washed extensively with PBST and endogenous peroxidase activity was quenched with a peroxidase suppressor. Detection was performed using a biotin-conjugated secondary antibody and SA-HRP followed by colorimetric detection using DAB. Tissues were counterstained with hematoxylin and prepped for mounting.

  • Immunohistochemistry was performed on normal biopsies of deparaffinized Rat cerebellum tissue. To expose target proteins heat induced antigen retrieval was performed using 10mM sodium citrate (pH6.0) buffer microwaved for 8-15 minutes. Following antigen retrieval tissues were blocked in 3% BSA-PBS for 30 minutes at room temperature. Tissues were then probed at a dilution of 1:20 with a mouse monoclonal antibody recognizing Calmodulin ab2860 or without primary antibody (negative control) overnight at 4°C in a humidified chamber. Tissues were washed extensively with PBST and endogenous peroxidase activity was quenched with a peroxidase suppressor. Detection was performed using a biotin-conjugated secondary antibody and SA-HRP followed by colorimetric detection using DAB. Tissues were counterstained with hematoxylin and prepped for mounting.

References

This product has been referenced in:
  • Li T  et al. The interactome and spatial redistribution feature of Ca2+receptor protein calmodulin reveals a novel role in invadopodia-mediated invasion. Cell Death Dis 9:292 (2018). Read more (PubMed: 29463791) »
  • Salman MM  et al. Hypothermia increases aquaporin 4 (AQP4) plasma membrane abundance in human primary cortical astrocytes via a calcium/transient receptor potential vanilloid 4 (TRPV4)- and calmodulin-mediated mechanism. Eur J Neurosci 46:2542-2547 (2017). ELISA . Read more (PubMed: 28925524) »
See all 6 Publications for this product

Customer reviews and Q&As

1-8 of 8 Abreviews or Q&A

Answer

Vielen Dank für Ihren Anruf und für Ihr Interesse an unseren Produkten.

Unter unseren anti-Calmodulin- Antikörpern ist ab2860 vermutlich der aussichtsreichste Kandidat: Das Immunogen (von Dictyostelium discoideum, SwissProtID P02599) weist eine Sequenzähnlichkeit von 83% gegen Calmodulin von Weizen (Triticum aestivum,P04464) auf. Daher ist eine Kreuzreaktivität von ab2860 sehr wahrscheinlich.

https://www.abcam.com/index.html?datasheet=2860 (or use the following: https://www.abcam.com/index.html?datasheet=2860).

ab2860 können wir für die Anwendungen ELISA, Western blot und Durchflusszytometrie mit diversen Spezies (Ratte, Huhn etc) garantieren. Unseres Wissens nach wurde der Antikörper aber bisher noch nicht in mit Weizen getestet. Falls Sie dies selbst testen möchten, kann ich Ihnen zur Zeit ein spezielles Angebot über einen 100%igen Abreview-Rabatt anbieten. Bei diesem Angebot bekommen Sie einen kostenlosen primären Antikörper, wenn Sie uns ein Abreview mit dem Testresultat zusenden. Um von diesem Angebot profitieren zu können, folgen Sie bitte diesen Schritten:

1.) Bestätigen Sie mir bitte, dass Sie ab2860 mit Weizen testen möchten.

2.) Bitte bestellen Sie erst nach Erhalt des Rabattcodes.

3.) Bestellen Sie den Antikörper wie üblich per Telefon, Email oder Fax

4.) Testen Sie den Antikörper mit Weizen.

5.) Teilen Sie uns das Ergebnis Ihres Tests durch ein Abreview mit und notieren Sie den Discount Code in dem Feld "additional notes". Unter der folgenden URL können Sie mehr über unser Abreview System erfahren: https://www.abcam.com/abreviews

6.) Der Rabattcode ist nach dem Abschicken des Abreviews aktiv, und Sie können einen anderen primären Antikörper bei uns bestellen (halten Sie bei der Bestellung bitte den Rabattcode und die Bestellnummer bereit). Bitte beachten Sie, dass der Rabattcode innerhalb von 4 Monaten nach Ausstellung eingelöst werden muss.

Der Rabattcode wird gültig unabhängig davon, ob Ihr Ergebnis positiv oder negativ ist.

Die Bedingungen zu unserem 100% Abreview Rabatt können Sie unter dem folgenden Link nachlesen: https://www.abcam.com/collaborationdiscount

Ich hoffe, diese Informationen helfen Ihnen weiter. Falls Sie einen Rabattcode erhalten möchten oder weitere Fragen haben, zögern Sie bitte nicht, sich wieder an mich zu wenden.

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Abcam has not validated the combination of species/application used in this Abreview.
Application
Western blot
Sample
Schizosaccharomyces pombe Recombinant protein (N/A)
Loading amount
10 µg
Specification
N/A
Gel Running Conditions
Reduced Denaturing (15%)
Blocking step
Milk as blocking agent for 30 minute(s) · Concentration: 3% · Temperature: 20°C

Abcam user community

Verified customer

Submitted Mar 09 2011

Answer

Thank you for your phone enquiry and questionaire. I'm sorry to hear you are having a problem with ab2860 (calmodulin Ab) in Western blotting. We have received other enquiries regarding this antibody and I would like to suggest the following modifications to your protocol: 1) Increase amount of protein loaded - try 50ug (others have even used 100ug) 2) Gel - increase gel percentage to 12-15%, this is better for smaller proteins; also, use PVDF membranes so smaller proteins won't run through 3) Blocking buffer - try straight milk in PBS and use straight PBS for washings (3X 5min) 4) Secondary Ab - does the product sheet suggest 1-15,000? Try 1:10,000. 5) Detection method - use ECL+, it's more sensitive than ECL. 6) Primary control - please include to know if weak signal is unspecific background staining. Please let me know if this helps, you can even e-mail me your blot! Do not hesitate to contact us for further advice.

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Answer

Thank you for your enquiry. I browsed the reference, Cell Mot. Cytoskel., 18: 113-122, 1991. There are a few suggestions, taken from the article, that I can give. These researchers had difficulty with their proteins transferring through the membrane. They recommended PVDF as the retention of calmodulin was better. If using nitrocellulose, you can place two pieces to prevent the small proteins from passing through the membrane and into the buffer. The originator has tested this product in BL21 (E.coli) cell lysates. They loaded 40-50 ug of protein from the cell lysate, an 18% gel was used and transfer conditions were with a semi-dry at 1 ampere for 1 hour. Normal immunoblotting parameters followed. The same was done with rat brain except 100 ug of protein was loaded. The originator has not tested this product in Chlamydomonas reinhardtii, the cross-reactivity information comes from a third party. I have enclosed a specific protocol below that might be of assistance. Please let me know if I can be of further assistance. Western blot detection of Calmodulin 1. Run samples on SDS/PAGE discontinuous gel (4% stacking gel, 15% running gel). Soak for 15 minutes in KP buffer (25 mM KH2/K2HPO4 buffer, pH 7.0). 2. Pre-wet Immobilon PVDF transfer membrane (Millpore) briefly in absolute methanol, and rinse for 15 minutes in KP buffer. 3. Transfer in KP buffer at 20 V overnight at 4°C. 4. Incubate membrane for 45 minutes at room temperature in 0.2% (v/v) glutaraldehyde freshly prepared in KP buffer. 5. Rinse in KP buffer and then block in 2% (w/v) BSA in TBS (20 mM Tris-HCl, 150 mM NaCl, pH 7.2) overnight at 4°C or 1 hour at 37°C. 6. Rinse the membrane in TBS containing 0.05% (v/v) Tween-20, 3 times for 10 minutes each at room temperature. (WASH) 7. Incubate with Anti-Calmodulin Antibody diluted in TBS for 1 hour at 37°C. 8. WASH. 9. Incubate with HRP-conjugated secondary, diluted in 2% BSA (w/v), 10% normal goat serum (v/v), 0.05 % Tween-20 (v/v), in TBS, for 1 hour at 37°C. 10. WASH. 11. Incubate membrane in AEC for up to 30 minutes at room temp for color development. AEC Solutions (Store at 4°C) Solution A: 0.17 g 3-amino-9-ethylcarbazole (AEC) in 10 ml dimethylformamide. Solution B: 0.47 g succinic acid, 2.38 g sodium acetate, 0.08 g thimerosal in dH2O to yield 20 ml final volume. Solution C: 3% (v/v) hydrogen peroxide in dH2O. Before use, mix 0.1 ml A, 0.2 ml B, 0.1 ml C in 9.6 ml dH2O. *Procedural details taken from D. Hulen, et. al. Cell Motility and the Cytoskeleton, 18: 113-122, 1991. The procedure listed above is intended only as a guide. The concentration of primary antibody listed here is a one that has been reported by users to have worked with the above procedure. Various assay conditions require that the optimal working concentrations be determined by serial dilution. The results that you obtain may vary depending on experimental conditions and technique.

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Question

BATCH NUMBER 130172 ORDER NUMBER 119538 DESCRIPTION OF THE PROBLEM No signal at all SAMPLE Tried 4 different species/cell extracts: CHO, HEK293, PC12 and DH5alpha bacteria (E.Coli). PRIMARY ANTIBODY Mouse mono to Calmodulin (ab2860-200) 1:500 as recommended, incubation at least 1hour, wash 4 times in PBS-0.5%tween for 10 min each with gentle rocking (also tried 1:1000) DETECTION METHOD ECL from Amersham POSITIVE AND NEGATIVE CONTROLS USED none ANTIBODY STORAGE CONDITIONS -20C, aliquoted, did not freeze/defreeze more than 2 times SAMPLE PREPARATION PC12(rat): lysed 2 confluent 10 cm plates in 500ul RIPA (1%NP40,0.1%deoxycholic acid, PBS pH 7.4, 1tablet prot.inh.) CHO and HEK293:lysed 2 confluent plates in 500 ul RIPA (0.5%NP40) DH5alpha: grow in 5 ml 2YT, spin, remove supt, lysed in 500ul GST lysis buffer(0.5%NP40, 1mMEDTA, 1 tablet prot.inh (Roche),PBS) NEVER heated any of the lysates above 4C. AMOUNT OF PROTEIN LOADED PC12 and DH5alpha: used 6X gel loading buffer,so I could load 40ul of lysate CHO and HEK293: loaded 25ul of lysate Did not measure the protein concentration, but this is what i load for other experiments and the other antibodies I use work fine. ELECTROPHORESIS/GEL CONDITIONS SDS-PAGE 10% TRANSFER AND BLOCKING CONDITIONS Transfer ON at 35 volts in Transfer buffer (57.7 g Glycine, 12 g Tris, 800 ml MeOH in 4L water) Blocking: 5% skim milk SECONDARY ANTIBODY HRP anti mouse, 1:5000, [ a competitor], at leas 1 hr in 5%milk-PBS-tween, wash 4 times in PBS-tween HAVE YOU RUN A "NO PRIMARY" CONTROL? No ADDITIONAL NOTES I have no signal at all even if I expose the film ON, I think that this batch of antibody might be bad. Maybe it does not react with human or mouse, but I don't see why it would not react with RAT or E.Coli. I don't know if it would work better at dilutions such as 1:200, but I think that this would be ridiculous and I would not want such a poor antibody anyway. If possible, we would like to be refunded, because we have wasted enough of time waiting for the antibody (3 weeks) and then testing it. We would like to simply buy it from another company.

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Answer

I'm very sorry to hear you are experiencing problems with ab2860. I would also expect the lysates in rat and E.coli samples to work with this antibody, however there may be several reasons for the lack of signal you are currently experiencing. The problem could be due to the incubation time which is only 1 hour. I would recommend to incubate overnight at 4C to give plenty of time for the antibody to bind to the membrane. Our recommendation of 1/500 was optimized on a blot of purified Dictyostelium calmodulin and your samples may need more concentrated antibody. Although you say that your samples should contain plenty of protein I would recommend to determine the exact protein concentration in your lysates and add 30-40ug lysate per well. I would also like to suggest using an ECl+ detection method as ECl is less sensitive, and to make sure the protease inhibitors in your samples are fresh. You mention having defrosted the antibody " was aliquoted, did not freeze/defreeze more than 2 times". We recommend to use a fresh aliquot every time as some antibodies do not tolerate re-freezing. You mention blocking in milk but do not mention how long for. In our experience milk can actually prevent binding of the antibody if applied too much; I would therefore recommend trying a one hour blocking stage and incubating the antibody in TBST only, and also to try 5%BSA 1hr. I hope you will find those recommendations helpful. If you still experience problems with those changes please do not hesitate to contact me again and I will arrange a refund if the antibody was purchased in the last 90days,

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Answer

Thank you for those extra details about your protocol this helped me a lot as I looked deeper into the cross reactivity of this antibody with your samples: 293T and HeLa cells are from human origin and 3T3 cells are from mouse origin. This could be the reason for the lack of signal, the antibody has only been tested on Cow, Chicken, E. coli, Rat, Chlamydomonas reinhardtii and Dictyostelium discoideum, and though I couldn't find the immunogen sequence ( Dictyostelium discoideum calmodulin) I checked the homology of other proteins which cross reacted with ab2860, to have an idea of the homology between the species: for example the chicken protein and the human protein homology was 87%, which is high but not an exact match, therefore this can explain the lack of signal. We only offer a refund for antibodies not working in applications and species tested as detailed on the datasheet so I cannot offer you a refund, however I would recommend running a positive control of Dictyostelium cell lysate and to try an anti-calmodulin antibody which is known to cross react with most mammals (e.g. ab1288), I hope this will help you, please do not hesitate to contact me again if you need further assistance,

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Answer

I'm sorry to hear you are having a problem with ab2860. We have not had a complaint about this antibody before. Thank you for taking the time to fill in our questionnaire, it helped me to understand your problem and it is useful to know that another antibody worked, pointing towards an incubation problem with ab2860 or a damaged antibody. Could you please clarify a few more details so I can understand even better your protocol: -what species are the samples -what blocking buffer was used and for how long -the dilution buffer used for ab2860 -the incubation time for ab2860 -have you tried a lower dilution? Our starting dilution of 1:500 is a recommendation and depending on the samples a final dilution may need to be used (eg 1:250-1:300)? Finally it would be good if I could find out your order details so I can look into possible delays in transport. I look forward to hearing from you to help you solve this matter, please be assured that should we find the antibody was at fault we will arrange a refund as requested, if you purchased the antibody in the last 90 days (I will need the order details to process the refund), My apologies for the delay,

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Answer

Thank you for your enquiry and for your patience. Regarding the specific epitope mapping, we have not performed these studies. Both immunogens were: Calmodulin purified from Dictyostelium discoideum and both antibodies have been tested for application in ELISA. However, that is all the information that we have at this time. These two antibodies are only available in the unconjugated form. If you have any more questions, please do contact us again.

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