Anti-Histone H3 (citrulline R2) antibody [EPR17703]

• I-ELISA

Supplier Data

Indirect ELISA - Anti-Histone H3 (citrulline R2) antibody [EPR17703] (AB176843)

ELISA analysis of Human Histone H3 (ci recombinant protein at 100 ng/ml with ab176843. An Alkaline Phosphatase-conjugated AffiniPure Goat Anti-Rabbit IgG (H+L) at 1/2500 dilution was used as the secondary antibody.

• IP

Supplier Data

Immunoprecipitation - Anti-Histone H3 (citrulline R2) antibody [EPR17703] (AB176843)

Histone H3 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 ?M lonomycin for 4 hours, whole cell lysate 10 ug with ab176843 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab176843 at 1/1000 dilution. VeriBlot for IP secondary antibody(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 ?M lonomycin for 4 hours, whole cell lysate 10 ug

Lane 2 : ab176843 IP in HEK-293T transfected with PADI4 expression vector containing a GFP-tag treated with 10 mM calcium chloride and 10 ?M lonomycin for 4 hours whole cell lysate

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab176843 in HEK-293T transfected with PADI4 expression vector containing a GFP-tag treated with 10 mM calcium chloride and 10 ?M lonomycin for 4 hours whole cell lysate

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

Exposure time : 3 min

All lanes:

Immunoprecipitation - Anti-Histone H3 (citrulline R2) antibody [EPR17703] (ab176843)

Predicted band size: 15 kDa

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• WB

Supplier Data

Western blot - Anti-Histone H3 (citrulline R2) antibody [EPR17703] (AB176843)

Blocking/Dilution buffer : 5% NFDM/TBST.

PADI4 catalyzes the citrullination of arginine residues of histone proteins.

All lanes:

Western blot - Anti-Histone H3 (citrulline R2) antibody [EPR17703] (ab176843) at 1/500 dilution

Lane 1:

NIH/3T3 (Mouse embyro fibroblast cells) cells transfected with vector control

Lane 2:

NIH/3T3 (Mouse embyro fibroblast cells) cells transfected with PADI4

Secondary

All lanes:

Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/1000 dilution

Predicted band size: 15 kDa

Observed band size: 15 kDa

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Exposure time: 30s

• WB

Supplier Data

Western blot - Anti-Histone H3 (citrulline R2) antibody [EPR17703] (AB176843)

Blocking/Dilution buffer : 5% NFDM/TBST.

PADI4 catalyzes the citrullination of arginine residues of histone proteins.

All lanes:

Western blot - Anti-Histone H3 (citrulline R2) antibody [EPR17703] (ab176843) at 1/500 dilution

Lane 1:

MCF7 (Human breast adenocarcinoma cell line) cells transfected with vector control

Lane 2:

MCF7 (Human breast adenocarcinoma cell line) cells transfected with PADI4

Secondary

All lanes:

Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/1000 dilution

Predicted band size: 15 kDa

Observed band size: 15 kDa

false

Exposure time: 30s

• PepArr

Unknown

Peptide Array - Anti-Histone H3 (citrulline R2) antibody [EPR17703] (AB176843)

ab176843 was 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, is available under the Product Protocol section.

• WB

CiteAb

Western blot - Anti-Histone H3 (citrulline R2) antibody [EPR17703] (AB176843)

Histone H3 (citrulline R2) western blot using anti-Histone H3 (citrulline R2) antibody [EPR17703] ab176843. Publication image and figure legend from Zhou, Y., Chen, B., et al., 2017, Front Immunol, PubMed 28993780.

ab176843 was used in this publication in western blot. This may not be the same as the application(s) guaranteed by Abcam. For a full list of applications guaranteed by Abcam for ab176843 please see the product overview.

Citrullination of extracellular substrates by intact neutrophils or neutrophil-conditioned media. (A) Upper panel, anti-citrullinated fibrinogen immunoblot of the supernatant from the incubation of fibrinogen for the indicated times in the presence of human neutrophils (lanes 1–7). Lane 7, fibrinogen was omitted. Lane 8 only contained fibrinogen. Lane 9, fibrinogen and recombinant PAD4. Lane 10, neutrophil-conditioned media. Lane 11, neutrophil with 2 mM EGTA. Lower panel, Coomassie Brilliant blue staining as a loading control. The bands corresponding to the α, β, and γ chains of fibrinogen are indicated. Note that α-fibrinogen is rapidly degraded by neutrophil-associated proteases. (B) Control immunoblot with anti-GAPDH, an intracellular protein, to demonstrate that significant lysis of neutrophils occurs only after 4 h of incubation with Triton X at 37°C under the experimental conditions. (C) Upper panel, anti-citrullinated histone H3 immunoblot of the supernatant from the incubation of histone H3 for the indicated time periods in the presence of human neutrophils (lanes 1–7). Lane 7, histone was omitted. Lane 8 only contained histone H3. Lane 9 contained histone H3 and recombinant PAD4. Lane 10, neutrophil-conditioned media. Lane 11, neutrophil with 2 mM EGTA. Note that citrullinated histone H3 is also cleaved by neutrophil-associated protease(s) to generate a slightly smaller protein. Lower panel, anti-histone H3 immunoblot as a loading control. This antibody does not recognize the proteolytically cleaved H3. (D) A similar experiment performed in the presence of an MMP inhibitor, a protease inhibitor cocktail, or both, as indicated. Lower panel, Coomassie Brilliant blue staining as a loading control. All data are representative of five independent experiments with different donors.

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