Recombinant Anti-IP10 antibody [EPR24674-12] (ab283681)

All lanes : Anti-IP10 antibody [EPR24674-12] (ab283681) at 1/1000 dilution

Lane 1 : Untreated Wild-type A549 (human lung carcinoma epithelial cell), whole cell lysate at 40 µg
Lane 2 : Wild-type A549 treated with 100 ng/ml IFN-y (ab259377) for 32 hours and 10 ng/m TNF-alpha (ab259410) for 32 hours, and 5ug/ml Brefeldin A (ab120299) for the last 6 hours, whole cell lysate at 40 µg
Lane 3 : Untreated IP10 knockout A549 whole cell lysate at 40 µg
Lane 4 : IP10 knockout A549 treated with 100 ng/ml IFN-y (ab259377) for 32 hours and 10 ng/m TNF-alpha (ab259410) for 32 hours, and 5ug/ml Brefeldin A (ab120299) for the last 6 hours, whole cell lysate at 40 µg
Lane 5 : Untreated THP-1 (human monocytic leukemia monocyte), whole cell lysate at 20 µg
Lane 6 : THP-1 treated with 200ng/ml IFN-y (ab259377) for 24 hours and 50ng/ml LPS for 24 hours, and 5ug/ml Brefeldin A for the last 21 hours, whole cell lysate at 20 µg

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (IRDye® 800CW) (ab216773) and Goat Anti-Mouse IgG H&L (IRDye® 680RD) (ab216776) at 1/10000 dilution

Predicted band size: 10 kDa
Observed band size: 11 kDa why is the actual band size different from the predicted?

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

Lanes 1-6: Merged signal (red and green). Green - ab283681 observed at 11 kDa. Red-loading control ab8245 (Mouse monoclonal [6C5] to GAPDH) was observed at 36 kDa.

ab283681 Anti-TNF Receptor I antibody [EPR24674-12] was shown to specifically react with IP10 in treated wild-type A549 cells. Loss of signal was observed when IP10 knockout cell lines ab266971 (knockout cell lysate ab256888) were used. Wild-type and IP10 knockout samples were subjected to SDS-PAGE. ab283681 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated at 4°C overnight at 1 in 1000 dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (ab216776) secondary antibodies at 1 in 10000 dilution for 1 hour at room temperature before imaging.

 

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized THP-1 cells labelling IP10 with ab283681 at 1/50 (12.86 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 (2 ug/ml) dilution (Green). Confocal image showing cytoplasmic staining in THP-1 cell line after treatment with Interferon gamma (200 ng/ml) and lipopolysaccharide (50 ng/ml) for 3 h, then adding Brefeldin A (1 ug/ml) for another 21 h. ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 (2.5 ug/ml) dilution (Red). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control: Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 (2 ug/ml) dilution.

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized CXCL10 KO A549 (ab266971) cells labelling IP10 with ab283681 at 1/50 (12.86 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 (2 ug/ml) dilution (Green). Confocal image showing the signal expression was increased in Parental A549 cells after treatment with Interferon gamma (200 ng/ml) and lipopolysaccharide (50 ng/ml) for 3h, then adding Brefeldin A (1 ug/ml) for another 21h, and no staining in treated CXCL10 KO A549 cells with the same conditions. ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 (2.5 ug/ml) dilution (Red). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control: Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 (2 ug/ml) dilution.