All lanes : Anti-IL-26 antibody [EPR22268-141] (ab224198) at 1/1000 dilution

Lane 1 : Untreated THP-1 (human monocytic leukemia cell line) whole cell lysate
Lane 2 : THP-1 treated with 80nM PMA (Phorbol-12-myristate-13-acetate) for 24 hours, then 1µg/ml LPS (lipopolysaccharides) for 24 hours, whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 100000 mg/ml

Predicted band size: 20 kDa
Observed band size: 27 kDa why is the actual band size different from the predicted?


Exposure time: 92 seconds

Blocking/Dilution buffer: 5% NFDM/TBST.

The molecular weight observed is consistent with what has been described in the literature (PMID:14764663).

This antibody reacts with an unidentifiable protein around 75 kDa.

Immunohistochemical analysis of paraffin-embedded human colon tissue labeling IL-26 with ab224198 at 1/1000 dilution, followed by Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on lymphocytes of human colon (PMID: 18483078, 28852311) is observed. Counterstained with hematoxylin. Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20mins. The section was incubated with ab224198 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized THP-1 (human monocytic leukemia cell line) cells labeling IL-26 with ab224198 at 1/100 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing cytoplasmic staining in THP-1 cell line treated with 80nM PMA (Phorbol-12-myristate-13-acetate) for 16h, then 100ng/ml LPS (lipopolysaccharide) for 3h and add 300ng/ml BFA (Brefeldin A) for 3h. The nuclear counterstain is DAPI (blue). Tubulin is detected with Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) (ab195889) at 1/200 dilution (red). Secondary antibody only control: Used PBS instead of primary antibody, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution.

Cells were either untreated, or treated with 80nM PMA (Phorbol-12-myristate-13-acetate) for 16h, then 100ng/ml LPS (lipopolysaccharide) for 3h and add 300ng/ml BFA (Brefeldin A) for 3h.

IL-26 was immunoprecipitated from 0.35 mg of THP-1 (human monocytic leukemia cell line) (treated with 80nM PMA (Phorbol-12-myristate-13-acetate) overnight, then 100ng/ml LPS (lipopolysaccharide) for 3h and add 300ng/ml BFA (Brefeldin A) for 3h) whole cell lysate with ab224198 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab224198 at 1/500 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used as secondary antibody at 1/1000 dilution.

Lane 1: THP-1 (treated as above) whole cell lysate 10 μg (Input).
Lane 2: ab224198 IP in THP-1 (treated as above) whole cell lysate.
Lane 3: : Rabbit monoclonal IgG (ab172730) instead of ab224198 in THP-1 (treated as above) whole cell lysate.

Blocking/Dilution buffer: 5% NFDM/TBST.
Exposure time: 3 mins.

Anti-IL-26 antibody [EPR22268-141] (ab224198) at 1/1000 dilution + His-tagged human IL-26 recombinant protein (aa 22-171), 20 ng

Secondary
Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 20 kDa


Exposure time: 26 seconds

Blocking/Dilution buffer: 5% NFDM/TBST.

Immunohistochemical analysis of paraffin-embedded human cervical carcinoma tissue labeling IL-26 with ab224198 at 1/1000 dilution, followed by Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on lymphocytes of human cervical carcinoma (PMID: 18483078, 28852311) is observed. Counterstained with hematoxylin. Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20mins. The section was incubated with ab224198 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument.