Ionomycin Ca2+ Salt, Ca2+ ionophore

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Ionomycin Ca2+ Salt, Ca2+ ionophore (AB120116)

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.

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Ionomycin Ca2+ Salt, Ca2+ ionophore (AB120116)

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.

• FuncS

Unknown

Functional Studies - Ionomycin Ca2+ Salt, Ca2+ ionophore (AB120116)

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.

• sELISA

Unknown

Sandwich ELISA - Ionomycin Ca2+ Salt, Ca2+ ionophore (AB120116)

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).

• IP

Lab

Immunoprecipitation - Ionomycin Ca2+ Salt, Ca2+ ionophore (AB120116)

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:

Immunoprecipitation - Anti-Histone H3 (citrulline R8) antibody [EPR20358-13] (<a href='/en-us/products/primary-antibodies/histone-h3-citrulline-r8-antibody-epr20358-13-ab219406'>ab219406</a>)

Predicted band size: 15 kDa

false

Exposure time: 1s

• IP

Lab

Immunoprecipitation - Ionomycin Ca2+ Salt, Ca2+ ionophore (AB120116)

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.

All lanes:

Immunoprecipitation - Anti-Histone H3 (citrulline R17) antibody [EPR20358-120] (<a href='/en-us/products/primary-antibodies/histone-h3-citrulline-r17-antibody-epr20358-120-ab219407'>ab219407</a>)

Predicted band size: 15 kDa

false

Exposure time: 1s

• WB

Supplier Data

Western blot - Ionomycin Ca2+ Salt, Ca2+ ionophore (AB120116)

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

The expression profile/ molecular weight observed is consistent with what has been described in the literature (PMID : 23129404 and PMID : 6423641).

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/200000 dilution.

All lanes:

Western blot - Anti-Interferon gamma antibody [RM1245] (<a href='/en-us/products/primary-antibodies/interferon-gamma-antibody-rm1245-ab322926'>ab322926</a>) at 1/1000 dilution

Lane 1:

Untreated No-GFP-CD16.NK-92 (CD16 ectopically expressed natural killer(NK-92®) cell with no GFP tag) whole cell lysate at 20 µg

Lane 2:

No-GFP-CD16.NK-92 treated with 80nM PMA(TPA) (<a href='/en-us/products/biochemicals/phorbol-12-myristate-13-acetate-pma-pkc-activator-ab120297'>ab120297</a>) and 3uM Ionomycin (ab120116) for 5 hours, 300 ng/ml BFA was added for last 4 hours whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Observed band size: 17 kDa,19 kDa,23 kDa,36 kDa

false

Exposure time: 92s

• Chemical Structure

Lab

Chemical Structure - Ionomycin Ca2+ Salt, Ca2+ ionophore (AB120116)

2D chemical structure image of ab120116, Ionomycin Ca2+ Salt, Ca2+ ionophore