Anti-PADI2 / PAD2 antibody

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-PADI2 / PAD2 antibody (AB16478)

ICC/IF image of ab16478 stained human Hek293 cells. The cells were PFA fixed (10 min), permabilised in 0.1% PBS-Tween (20 min) and incubated with the antibody (ab16478, 5µg/ml) for 1h at room temperature. 1%BSA / 10% normal goat serum / 0.3M glycine was used to block non-specific protein-protein interactions. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red). DAPI was used to stain the cell nuclei (blue).

• WB

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Western blot - Anti-PADI2 / PAD2 antibody (AB16478)

This blot was produced using a 10% Bis-tris gel under the MOPS buffer system. The gel was run at 200V for 50 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using 5% Bovine Serum Albumin before being incubated with ab16478 overnight at 4°C. Antibody binding was detected using an anti-rabbit antibody conjugated to HRP, and visualised using ECL development solution. The 75-kDa band observed is consistent with what has been described in the literature (PMID : 18668562; 20668670; 16723463).+--+ | | +--+

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Western blot - Anti-PADI2 / PAD2 antibody (ab16478) at 1 µg/mL

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Human kidney tissue lysate - total protein (<a href='/en-us/products/unavailable/human-kidney-tissue-lysate-total-protein-ab30203'>ab30203</a>) at 20 µg

Secondary

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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 1/10000 dilution

Predicted band size: 76 kDa

Observed band size: 34 kDa,75 kDa

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Exposure time: 20min

• WB

CiteAb

Western blot - Anti-PADI2 / PAD2 antibody (AB16478)

PADI2 / PAD2 western blot using anti-PADI2 / PAD2 antibody ab16478. Publication image and figure legend from Zhou, Y., Chen, B., et al., 2017, Front Immunol, PubMed 28993780.

ab16478 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 ab16478 please see the product overview.

Western blot analysis of secreted PADs in neutrophil-conditioned media and determination of neutrophil PAD2/PAD4 extracellular activity. (A) Anti-PAD2 immunoblot of concentrated neutrophil-conditioned media from four different healthy donors (D1–D4). (B) Anti-PAD4 immunoblot of same samples with longer exposure time. (C) Characterization of the blocking anti-PAD2 and anti-PAD4 antibodies. Upper panel, anti-citrullinated histone H3 immunoblot of a reaction with recombinant PAD2 in the presence of control NIP228 IgG antibody (lane 1), blocking anti-PAD4 antibody (lanes 2–4), or a blocking anti-PAD2 antibody (lanes 5–7) at the indicated concentrations. Lower panel, similar reaction with recombinant PAD4 in the presence of the same antibodies. (D) Anti-citrullinated histone H3 immunoblot of the culture supernatant of human neutrophils incubated with histone H3 plus blocking anti-PAD2 or anti-PAD4 antibodies as indicated (lane 1–5); 2 mM EDTA was added to demonstrate the calcium-dependence of the reaction (lane 6); neutrophil-conditioned media, plus blocking antibodies as indicated (lanes 7–11). No antibodies were added in lanes 1 and 7, whereas control IgG NIP228 was added in lanes 2 and 8. (E) Anti-citrullinated fibrinogen immunoblot of the supernatant from the incubation of fibrinogen with neutrophils plus blocking antibodies. No antibodies were added in lane 1, whereas control IgG NIP228 was added in lane 2. 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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