Overview

  • Product name
    Ionomycin Ca2+ Salt, Ca2+ ionophore
  • Description
    Ca2+ ionophore
  • Biological description
    Ca2+ ionophore. Useful when calcium-dose response data are not required. Ion specificity Mn2+>Ca2+>Mg2+>Sr2+>Ba2+.
  • Purity
    > 99%
  • CAS Number
    56092-82-1
  • Chemical structure
    Chemical Structure

Properties

Applications

Our Abpromise guarantee covers the use of ab120116 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
Functional Studies Use at an assay dependent concentration.

Images

  • All lanes : Anti-IL-17A antibody [EPR21776] (ab218013) at 1/1000 dilution

    Lane 1 : Untreated EL4 (mouse lymphoma T lymphocyte) whole cell lysate
    Lane 2 : EL4 treated with 50 ng/ml Phorbol-12-myristate-13-acetate (PMA) and 500 ng/ml ionomycin (ab120116) for 6 hours, then with 500 ng/ml Brefeldin A for 18 hours, whole cell lysate

    Lysates/proteins at 10 µg per lane.

    Secondary
    All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

    Developed using the ECL technique.

    Observed band size: 14,17 kDa
    why is the actual band size different from the predicted?


    Exposure time: 3 minutes


    Blocking/dilution buffer and concentration: 5% NFDM/TBST

    Expression of IL-17A can be induced by PMA and Ionomycin treatment (PMID 28382171).

    The expression profile is consistent with the literature (PMID 9764847).

  • All lanes : Anti-Histone H3 (citrulline R8) antibody [EPR20358-13] (ab219406) at 1/10000 dilution

    Lane 1 : Whole cell lysate from HEK-293T (Human epithelial cell line from embryonic kidney) transfected with empty vector with GFP tag (vector control), then treated with 10mM CaCl2 and 10µM Ionomycin (ab120116) for 2 hours
    Lane 2 : Whole cell lysate from HEK-293T cells transfected with PADI4 (WT), then treated with 10mM CaCl2 and 10µM Ionomycin (ab120116) for 2 hours

    Lysates/proteins at 10 µg per lane.

    Secondary
    All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

    Observed band size: 15 kDa why is the actual band size different from the predicted?


    Exposure time: 1 second


    Blocking/Dilution buffer: 5% BSA/TBST.

    Histone H3R8 is citrullinated by PADI4 and CaCl2 is used as a cofactor according to the literature (PMID: 16567635). Ionomycin is used to improve the modification by PADI4 according to the literature (PMID: 26360112).

  • All lanes : Anti-Histone H3 (citrulline R8) antibody [EPR20358-13] (ab219406) at 1/5000 dilution

    Lane 1 : Whole cell lysate from NIH/3T3 (Mouse embryonic fibroblast cell line) transfected with empty vector with GFP tag (vector control), then treated with 10mM CaCl2 for 2 hours
    Lane 2 : Whole cell lysate from NIH/3T3 transfected with PADI4 (WT) then treated with 10mM CaCl2 for 2 hours
    Lane 3 : Whole cell lysate from C6 (Rat glial tumor cell line) transfected with empty vector with GFP tag (vector control) then treated with 10mM CaCl2 and 10µM Ionomycin (ab120116) for 2 hours
    Lane 4 : C6 transfected with PADI4 (WT), then treated with 10mM CaCl2 and 10µM Ionomycin (ab120116) for 2 hours

    Lysates/proteins at 10 µg per lane.

    Secondary
    All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

    Observed band size: 15 kDa why is the actual band size different from the predicted?


    Exposure time: 1 second


    Blocking/Dilution buffer: 5% BSA/TBST.

  • Flow cytometric analysis of 4% paraformaldehyde-fixed HEK-293T (Human epithelial cell line from embryonic kidney) transfected with empty vector (left panel) or PADI4 (WT, right panel), then treated with 10mM CaCl2 and 10μM Ionomycin (ab120116) for 2 hours, labeling Histone H3 (citrulline R8) with ab219406 at 1/500 dilution, followed by Goat anti rabbit IgG (Alexa Fluor® 647) at 1/2000 dilution.

    Positive signal is obtained from HEK-293T cells transfected with WT PADI4 treated with 10mM CaCl2 and 10μM Ionomycin (ab120116) for 2 hours.

  • Histone H3 (citrulline R8) was immunoprecipitated from 0.35 mg of HEK-293T (Human epithelial cell line from embryonic kidney) transfected with PADI4 (WT), then treated with 10mM CaCl2 and 10μM Ionomycin (ab120116) for 2 hours, whole cell lysate with ab219406 at 1/30 dilution.

    Western blot was performed from the immunoprecipitate using ab219406 at 1/1000 dilution.

    VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10000 dilution.

    Lane 1: HEK-293T transfected with PADI4 (WT), then treated with 10mM CaCl2 and 10μM Ionomycin (ab120116) for 2 hours, whole cell lysate 10 µg (Input).
    Lane 2: ab219406 IP in HEK-293T transfected with PADI4 (WT), then treated with 10mM CaCl2 and 10μM Ionomycin (ab120116) for 2 hours, whole cell lysate.
    Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab219406 in HEK-293T transfected with PADI4 (WT), then treated with 10mM CaCl2 and 10μM Ionomycin (ab120116) for 2 hours, whole cell lysate.

    Blocking and dilution buffer: 5% NFDM/TBST.

  • All lanes : Anti-Histone H3 (citrulline R17) antibody [EPR20358-120] (ab219407) at 1/5000 dilution

    Lane 1 : HEK-293T (Human epithelial cell line from embryonic kidney) transfected with empty vector with GFP tag (vector control), then treated with 10mM CaCl2 and 10µM Ionomycin (ab120116) for 2 hours, whole cell lysate
    Lane 2 : HEK-293T (Human epithelial cell line from embryonic kidney) transfected with PADI4 (WT), then treated with 10mM CaCl2 and 10µM Ionomycin (ab120116) for 2 hours, whole cell lysate

    Lysates/proteins at 10 µg per lane.

    Secondary
    All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

    Observed band size: 15 kDa why is the actual band size different from the predicted?


    Exposure time: 3 seconds


    Blocking/Dilution buffer: 5% BSA/TBST.

    Histone H3R17 is citrullinated by PADI4 and CaCl2 is used as a cofactor according to the literature (PMID: 16567635). Ionomycin is used to improve the modification by PADI4 according to the literature (PMID: 26360112).

  • All lanes : Anti-Histone H3 (citrulline R17) antibody [EPR20358-120] (ab219407) at 1/5000 dilution

    Lane 1 : NIH/3T3 (Mouse embryonic fibroblast cell line) transfected with empty vector with GFP tag (vector control), then treated with 10mM CaCl2 for 2 hours, whole cell lysate
    Lane 2 : NIH/3T3 (Mouse embryonic fibroblast cell line) transfected with PADI4 (WT) then treated with 10mM CaCl2 for 2 hours, whole cell lysate
    Lane 3 : C6 (Rat glial tumor cell line) transfected with empty vector with GFP tag (vector control) then treated with 10mM CaCl2 and 10µM Ionomycin (ab120116) for 2 hours, whole cell lysate
    Lane 4 : C6 (Rat glial tumor cell line) transfected with PADI4 (WT), then treated with 10mM CaCl2 and 10µM Ionomycin (ab120116) for 2 hours, whole cell lysate

    Lysates/proteins at 10 µg per lane.

    Secondary
    All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

    Observed band size: 15 kDa why is the actual band size different from the predicted?


    Exposure time: 1 second


    Blocking/Dilution buffer: 5% BSA/TBST.

  • Flow cytometric analysis of 4% paraformaldehyde-fixed HEK-293T (Human epithelial cell line from embryonic kidney) transfected with empty vector (left panel) or PADI4 (WT, right panel), then treated with 10mM CaCl2 and 10μM Ionomycin (ab120116) for 2 hours, labeling Histone H3 (citrulline R17) with ab219407 at 1/500 dilution, followed by Goat anti rabbit IgG (Alexa Fluor® 647) at 1/2000 dilution.

    Positive signal is obtained from HEK-293T cells transfected with WT PADI4 treated with 10mM CaCl2 and 10μM Ionomycin (ab120116) for 2 hours.

  • Histone H3 (citrulline R17) was immunoprecipitated from 0.35 mg of HEK-293T (Human epithelial cell line from embryonic kidney) transfected with PADI4 (WT), then treated with 10mM CaCl2 and 10μM Ionomycin (ab120116) for 2 hours, whole cell lysate with ab219407 at 1/30 dilution.

    Western blot was performed from the immunoprecipitate using ab219407 at 1/1000 dilution.

    VeriBlot for IP secondary antibody (HRP) (ab131366), was used for detection at 1/10000 dilution.

    Lane 1: HEK-293T transfected with PADI4 (WT), then treated with 10mM CaCl2 and 10μM Ionomycin (ab120116) for 2 hours, whole cell lysate 10 µg (Input).
    Lane 2: ab219407 IP in HEK-293T transfected with PADI4 (WT), then treated with 10mM CaCl2 and 10μM Ionomycin (ab120116) for 2 hours, whole cell lysate.
    Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab219407 in HEK-293T transfected with PADI4 (WT), then treated with 10mM CaCl2 and 10μM Ionomycin (ab120116) for 2 hours, whole cell lysate.

    Blocking/Dilution buffer: 5% NFDM/TBST.

  • ab58668 staining ATF3 in A549 cells treated with ionomycin Ca2+ salt (ab120116), by ICC/IF. Increase in ATF3 expression correlates with increased concentration of ionomycin Ca2+ salt, as described in literature.
    The cells were incubated at 37°C for 2h in media containing different concentrations of ab120116 (ionomycin Ca2+ salt) in DMSO, fixed with 4% formaldehyde for 10 minutes at room temperature and blocked with PBS containing 10% goat serum, 0.3 M glycine, 1% BSA and 0.1% tween for 2h at room temperature. Staining of the treated cells with ab58668 (10 µg/ml) was performed overnight at 4°C in PBS containing 1% BSA and 0.1% tween. A DyLight 488 goat anti-mouse polyclonal antibody (ab96879) at 1/250 dilution was used as the secondary antibody. Nuclei were counterstained with DAPI and are shown in blue.

  • Sandwich ELISA - IFN gamma Human ELISA Kit (ab46025)

    Jurkat were stimulated for 48 hours with 50 ng x mL-1 of PMA (ab120297) and 1 uM Ionomycin (ab120116) and PBMCs were stimulated for 48 hours with 2 % PHA-M (LifeTechnologies). Cell free supernatants were tested, showing results after background signal was subtracted (duplicates +/- SD).

References

This product has been referenced in:
See all 4 Publications for this product

Customer reviews and Q&As

1-2 of 2 Abreviews or Q&A

Answer

Thank you for contacting us.

Product ab120116 (Ionomycin Ca2+ Salt) differs from ab120370 (free acid), as the presence of the Ca2+ makes the compound easier to solubilise (the free acid is soluble in ethanol to 10 mM and in DMSO to 10 mM. The Ca2+ salt is soluble in DMSO to 25 mM and in ethanol to 100 mM).

Apart from this, it may depend on your specific experimental requirements. Some researches prefer not to use the Ca2+ salt as they prefer not to introduce extra calcium into the system.

I hope this information was helpful. Please do not hesitate to contact us should you require further assistance.

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Question
Answer

Vielen Dank für Ihre Anfrage und dafür, dass Sie sich die Zeit genommen haben, unseren Fragebogen auszufüllen.

Die Bande bei 50 kDa sieht in der Tat unspezifisch aus; dies wäre allerdings ja nicht weiter schlimm, wenn Sie die spezifische Bande bei 120/140 kDa detektieren würden. Ihr Protokoll sieht absolut in Ordnung aus, und daher macht es mir etwas Sorgen, dass Sie keine positiven Ergebnisse erzielen.

Um der Sache auf den Grund zu gehen, würde ich Sie gerne noch nach einigen Details fragen:

Wie haben Sie die Jurkat-Zellen infiziert (Vpr-/wt/M)? Und wissen Sie, ob diese Infektion den T-Zellrezeptorkomplex aktiviert und damit die Expression von NFAT induziert?

Oder anders gefragt: Haben Sie eine Phorbol 12-myristate 13-acetate (PMA)/Ionomycin-stimulierte Positivkontrolle mitgeführt? Sollte der Antikörper mit dieser Kontrolle auch keine Bande für NFAT ergeben, wäre das der Beweis, dass mit dem Vial etwas nicht stimmt, und ich könnte Ihnen umgehend einen kostenlosen Ersatz zusenden. Wir haben zum einen PMA und Ionomycin in unserem Katalog, falls Sie Ihre Zellen selbst stimulieren möchten (ab120297, ab120116, ab120370), oder wir haben Lysate stimulierter Zellen wie zum Beispiel ab14850 oder ab14843 verfügbar.

Click here (or use the following: https://www.abcam.com/index.html?datasheet=120297).
Click here (or use the following: https://www.abcam.com/index.html?datasheet=120116).
Click here (or use the following: https://www.abcam.com/index.html?datasheet=120370).
Click here (or use the following: https://www.abcam.com/index.html?datasheet=14850).
Click here (or use the following: https://www.abcam.com/index.html?datasheet=14843).

Vielen Dank für Ihre Kooperation und Hilfsbereitschaft. Ich freue mich auf Ihre Antworten.

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Please note: All products are "FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES, NOT FOR USE IN HUMANS"
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