Anti-Histone H3 (citrulline R2 + R8 + R17) antibody [RM1001]

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-Histone H3 (citrulline R2 + R8 + R17) antibody [RM1001] (AB281584)

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 100% methanol permeabilized 293T (human embryonic kidney epithelial cell) cells labelling Histone H3 (citrulline R2 + R8 + R17) with primary antibody anti-Histone H3 (citrulline R2 + R8 + R17) (ab281584) at 1/2000 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor® 594) (ab150080) secondary antibody at 1/1000 dilution. Confocal image showing nuclear staining on 293T cells transfected with a human PADI4 expression vector containing a GFP tag, then treated with 10 mM CaCl2 and 10 μM ionomycin for 2 hours. The nuclear counter stain is DAPI (blue).

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-Histone H3 (citrulline R2 + R8 + R17) antibody [RM1001] (AB281584)

Intracellular Flow Cytometry analysis of 293T (human embryonic kidney epithelial cell) transfected with myc-tagged PADI4 construct, then treated with 10mM CaCl2 and 10uM Ionomycin for 4h labeling Histone H3 (citrulline R2 + R8 + R17) with ab281584 at 1/500 dilution. Cells were fixed with 4% paraformaldehyde and permeabilised with 90% methanol. Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) at 1/2000 dilution was used as a secondary antibody. Isotype control : Rabbit monoclonal IgG (ab172730)/left.

• IP

Supplier Data

Immunoprecipitation - Anti-Histone H3 (citrulline R2 + R8 + R17) antibody [RM1001] (AB281584)

Histone H3 (citrulline R2 + R8 + R17) was immunoprecipitated from 0.35 mg HEK-293T (human embryonic kidney) transfected with PADI4 expression vector containing a GFP-tag treated with 10 mM calcium chloride and 10 μM lonomycin for 4 hours, whole cell lysate 10 μg with 281584 at 1/30 dilution (2μg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using 281584 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP)(ab131366) was used at 1/5000 dilution.

Lane 1 : HEK-293T (human embryonic kidney) transfected with PADI4 expression vector containing a GFP-tag treated with 10 mM calcium chloride and 10 μM lonomycin for 4 hours, whole cell lysate 10 μg.

Lane 2 : ab281584 IP in HEK-293T transfected with PADI4 expression vector containing a GFP-tag treated with 10 mM calcium chloride and 10 μM lonomycin for 4 hours whole cell lysate

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab281584 in HEK-293T transfected with PADI4 expression vector containing a GFP-tag treated with 10 mM calcium chloride and 10 μM lonomycin for 4 hours whole cell lysate

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

Exposure time : 10 seconds

All lanes:

Immunoprecipitation - Anti-Histone H3 (citrulline R2 + R8 + R17) antibody [RM1001] (ab281584)

Predicted band size: 15 kDa

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• WB

Lab

Western blot - Anti-Histone H3 (citrulline R2 + R8 + R17) antibody [RM1001] (AB281584)

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

Exposure time : 180 seconds

All lanes:

Western blot - Anti-Histone H3 (citrulline R2 + R8 + R17) antibody [RM1001] (ab281584) at 1/1000 dilution

Lane 1:

Untreated C6 (rat glial tumor glial cell) transfected with PADI4 expression vector containing a GFP-tag, whole cell lysate at 20 µg

Lane 2:

C6 transfected with PADI4 expression vector containing a GFP-tag treated with 10 mM calcium chloride and 10 μM lonomycin for 2 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 1/100000 dilution

Predicted band size: 15 kDa

false

• WB

Lab

Western blot - Anti-Histone H3 (citrulline R2 + R8 + R17) antibody [RM1001] (AB281584)

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

Exposure time : 48 seconds

All lanes:

Western blot - Anti-Histone H3 (citrulline R2 + R8 + R17) antibody [RM1001] (ab281584) at 1/1000 dilution

Lane 1:

Untreated HEK-293T (human embryonic kidney) transfected with PADI4 expression vector containing a myc-His-tag®, whole cell lysate at 20 µg

Lane 2:

HEK-293T transfected with PADI4 expression vector containing a myc-His-tag® treated with 10 mM calcium chloride and 10 μM lonomycin for 4 hours, whole cell lysate at 20 µg

Lane 3:

Untreated NIH/3T3 (mouse embryonic fibroblast) transfected with PADI4 expression vector containing a myc-His-tag®, whole cell lysate at 20 µg

Lane 4:

NIH/3T3 transfected with PADI4 expression vector containing a myc-His-tag® treated with 10 mM calcium chloride and 10 μM lonomycin for 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 1/100000 dilution

Predicted band size: 15 kDa

false

• Dot

Supplier Data

Dot Blot - Anti-Histone H3 (citrulline R2 + R8 + R17) antibody [RM1001] (AB281584)

Dot blot analysis of Histone H3 (citrulline R2 + R8 + R17) using 281584 at 1/1000 (0.478 ug/ml) followed by a Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1/100,000 dilution.

Lane 1 : Histone H3 (citrulline R2) peptide (aa 1-21)

Lane 2 : Histone H3 (citrulline R8) peptide (aa 1-21)

Lane 3 : Histone H3 (citrulline R17) peptide (aa 1-21)

Lane 4 : Histone H3 (citrulline R2/R8/R17) peptide (aa 1-21)

Lane 5 : Unmodified histone H3 peptide (aa 1-21)

Exposure time : 3 minutes

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

• PepArr

Supplier Data

Peptide Array - Anti-Histone H3 (citrulline R2 + R8 + R17) antibody [RM1001] (AB281584)

Peptide array analysis of ab281584 at (0.1ug/ml) followed by a Goat Anti-Rabbit IgG, (H+L), Fluo 647nm conjugated at 1 : 50,000 dilution. All batches of ab281584 are tested in Peptide Array against 501 different modified and unmodified histone peptides; each peptide is printed on the array at six concentrations (each in triplicate). Circle area represents affinity between the antibody and a peptide : all antigen-containing peptides are displayed as red circles, all other peptides as blue circles. The affinity is calculated as area under curve when antibody binding values are plotted against the corresponding peptide concentration. Each circle area is normalized to the peptide with the strongest affinity. The complete dataset, including full list of all peptides and information on the position of each peptide in the diagram, can be downloaded from the 'documents' section.