Anti-PADI2 / PAD2 antibody (ab16478)

Image courtesy of Human Protein Atlas

ab16478 staining PADI2 in female rectum, showing a distinct and strong staining pattern in glandular cells. Paraffin embedded human rectal tissue was incubated with ab16478 (1/100 dilution) for 30 mins at room temperature. Antigen retrieval was performed by heat induction in citrate buffer pH 6.

ab16478 was tested in a tissue microarray (TMA) containing a wide range of normal and cancer tissues as well as a cell microarray consisting of a range of commonly used, well characterised human cell lines. Further results for this antibody can be found at www.proteinatlas.org

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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).

ab16478 staining human peripheral blood mononuclear cells (cultured with M-CSF) by Flow Cytometery. Cells were treated with flow cytometery staining buffer (PBS 0.1% sodium azide 1% BSA) and gating was done on myeloid cells. The primary antibody was diluted 1/10 (PBS 0.1% sodium azide 1% BSA) and incubated with sample for 20 minutes at 25°C. An Alexa Fluor® conjugated goat polyclonal to rabbit IgG was used undiluted as secondary.

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Anti-PADI2 / PAD2 antibody (ab16478) at 1 µg/ml + Human kidney tissue lysate - total protein (ab30203) at 20 µg

Secondary
Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/10000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 76 kDa
Observed band size: 75 kDa (why is the actual band size different from the predicted?)
Additional bands at: 34 kDa. We are unsure as to the identity of these extra bands.


Exposure time: 20 minutes

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).