Anti-Histone H3 (citrulline R17) antibody [EPR20358-120]

8 product images

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  Indirect ELISA - Anti-Histone H3 (citrulline R17) antibody [EPR20358-120] (ab219407)

  ELISA analysis of Human Histone H3 (citrulline R17) recombinant protein at 100 ng/ml with ab219407. An Alkaline Phosphatase-conjugated AffiniPure Goat Anti-Rabbit IgG (H+L) at 1/2500 dilution was used as the secondary antibody.

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  Immunocytochemistry/ Immunofluorescence - Anti-Histone H3 (citrulline R17) antibody [EPR20358-120] (ab219407)

  Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HEK-293T (Human epithelial cell line from embryonic kidney) cells transfected with GFP only or a GFP-tagged PADI4 expression construct, then treated with 10mM CaCl₂ for 2 hours, labeling Histone H3 (citrulline R17) with ab219407 at 1/100 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor^® 488) (Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077) secondary antibody at 1/1000 dilution (green).
  

  Confocal image showing positive staining on HEK-293T cells transfected with a GFP-tagged PADI4 expression construct, then treated with 10mM CaCl₂ for 2 hours.

  The nuclear counterstain is DAPI (blue).

  Tubulin is detected with Alexa Fluor® 594 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker ab195889 (Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594)) at 1/200 dilution (red).

  Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat anti-rabbit IgG (Alexa Fluor^® 488) (Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077) at 1/1000 dilution.

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  Immunoprecipitation - Anti-Histone H3 (citrulline R17) antibody [EPR20358-120] (ab219407)

  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 (Ionomycin Ca2+ Salt, Ca2+ ionophore 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 Detection Reagent (HRP) (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 (Ionomycin Ca2+ Salt, Ca2+ ionophore 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 (Ionomycin Ca2+ Salt, Ca2+ ionophore ab120116) for 2 hours, whole cell lysate.
  Lane 3: Rabbit monoclonal IgG (Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730) instead of ab219407 in HEK-293T transfected with PADI4 (WT), then treated with 10mM CaCl₂ and 10μM Ionomycin (Ionomycin Ca2+ Salt, Ca2+ ionophore ab120116) for 2 hours, whole cell lysate.

  Blocking/Dilution buffer: 5% NFDM/TBST.

  All lanes: Immunoprecipitation - Anti-Histone H3 (citrulline R17) antibody [EPR20358-120] (ab219407)

  Predicted band size: 15 kDa

  Exposure time: 1s

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  Western blot - Anti-Histone H3 (citrulline R17) antibody [EPR20358-120] (ab219407)

  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: Western blot - 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 (Ionomycin Ca2+ Salt, Ca2+ ionophore ab120116) for 2 hour at 10 µg

  Lane 2: HEK-293T (Human epithelial cell line from embryonic kidney) transfected with PADI4 (WT), then treated with 10mM CaCl2 and 10µM Ionomycin (Ionomycin Ca2+ Salt, Ca2+ ionophore ab120116) for 2 hours, whole cell lysate at 10 µg

  Secondary

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

  Predicted band size: 15 kDa

  Observed band size: 15 kDa

  Exposure time: 3s

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  Western blot - Anti-Histone H3 (citrulline R17) antibody [EPR20358-120] (ab219407)

  Blocking/Dilution buffer: 5% BSA/TBST.
  

  Exposure time: Lane 1: 1 second; Lane 2: 3 minutes.

  Lane 1: Western blot - Anti-Histone H3 (citrulline R17) antibody [EPR20358-120] (ab219407) at 1/1000 dilution

  Lane 2: Western blot - Anti-Histone H3 (citrulline R17) antibody [EPR20358-120] (ab219407) at 1/200 dilution

  Lane 1: E12 mouse embryo lysate at 10 µg

  Lane 2: E12 rat embryo lysate at 10 µg

  Secondary

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

  Predicted band size: 15 kDa

  Observed band size: 15 kDa

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  Western blot - Anti-Histone H3 (citrulline R17) antibody [EPR20358-120] (ab219407)

  Blocking/Dilution buffer: 5% BSA/TBST.

  All lanes: Western blot - 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 at 10 µg

  Lane 2: NIH/3T3 (Mouse embryonic fibroblast cell line) transfected with PADI4 (WT) then treated with 10mM CaCl2 for 2 hours, whole cell lysate at 10 µg

  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 (Ionomycin Ca2+ Salt, Ca2+ ionophore ab120116) for 2 hours, whole cell lysate at 10 µg

  Lane 4: C6 (Rat glial tumor cell line) transfected with PADI4 (WT), then treated with 10mM CaCl2 and 10µM Ionomycin (Ionomycin Ca2+ Salt, Ca2+ ionophore ab120116) for 2 hours, whole cell lysate at 10 µg

  Secondary

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

  Predicted band size: 15 kDa

  Observed band size: 15 kDa

  Exposure time: 1s

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  ELISA - Anti-Histone H3 (citrulline R17) antibody [EPR20358-120] (ab219407)

  Direct ELISA using ab219407 at 0-1000 ng/ml, followed by Alkaline Phosphatase-conjugated AffiniPure Goat Anti-Rabbit IgG(H+L) at 1/2500 dilution.
  

  Antigen concentration: 1000 ng/ml.

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  Flow Cytometry (Intracellular) - Anti-Histone H3 (citrulline R17) antibody [EPR20358-120] (ab219407)

  Intracellular 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 (Ionomycin Ca2+ Salt, Ca2+ ionophore 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 (Ionomycin Ca2+ Salt, Ca2+ ionophore ab120116) for 2 hours.