Recombinant Anti-IP10 antibody [EPR20764] (ab214668)

All lanes : Anti-IP10 antibody [EPR20764] (ab214668) at 1/1000 dilution

Lane 1 : Wild-type THP-1 vehicle control IFNg (0 ng/ml, 32 h), TNF-alpha (0 ng/ml, 32 h), Brefeldin A (5 ug/ml, 6 h) cell lysate
Lane 2 : Wild-type THP-1 treated IFNg (100 ng/ml, 32 h), TNF-alpha (10 ng/ml, 32 h), Brefeldin A (5 ug/ml, 6 h) cell lysate
Lane 3 : CXCL10 knockout THP-1 vehicle control IFNg (0 ng/ml, 32 h), TNF-alpha (0 ng/ml, 32 h), Brefeldin A (5 ug/ml, 6 h) cell lysate
Lane 4 : CXCL10 knockout THP-1 treated IFN-gamma (100 ng/ml, 32 h), TNF-alpha (10 ng/ml, 32 h), Brefeldin A (5 ug/ml, 6 h) cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

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

False colour image of Western blot: Anti-IP10 antibody [EPR20764] staining at 1/1000 dilution, shown in green; Mouse anti-Alpha Tubulin [DM1A] (ab7291) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab214668 was shown to bind specifically to IP10. A band was observed at 11 kDa in treated wild-type THP-1 cell lysates with no signal observed at this size in treated CXCL10 knockout cell line ab277860 (knockout cell lysate ab282997). To generate this image, wild-type and CXCL10 knockout THP-1 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween^® 20 (TBS-T) before incubation with primary antibodies overnight at 4°C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) at 1/20000 dilution.

All lanes : Anti-IP10 antibody [EPR20764] (ab214668) at 1/1000 dilution

Lane 1 : Wild-type A549 Brefeldin A (ab120299)-treated (5ug/ml, 6h) cell lysate
Lane 2 : Wild-type A549 IFN-y (ab259377) (100 ng/ml, 32 h) and TNF-alpha (ab259410) (10 ng/ml, 32h), and Brefeldin A (ab120299)-treated (5ug/ml for the last 6h) cell lysate
Lane 3 : IP10 knockout A549 Brefeldin A (ab120299)-treated (5ug/ml, 6h) cell lysate
Lane 4 : IP10 knockout A549 IFN-y (ab259377) (100ng/ml, 32h) and TNF-alpha (ab259410) (10ng/ml, 32h), and Brefeldin A (ab120299)-treated (5ug/ml for the last 6h) cell lysate
Lane 5 : THP-1 Brefeldin A (ab120299)-treated (5ug/ml, 6h) cell lysate
Lane 6 : THP-1 IFN-y (ab259377) (200ng/ml, 24h) and LPS (50ng/ml, 24h)-treated for 24 hours, and Brefeldin A (ab120299)-treated (5ug/ml for the last 6h) cell lysate

Lysates/proteins at 30 µg per lane.

Performed under reducing conditions.

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

Lanes 1 - 6: Merged signal (red and green). Green - ab214668 observed at 11 kDa. Red - loading control ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37kDa.

ab214668 was shown to react with IP10 in wild-type A549 cells in western blot with loss of signal observed in IP10 knockout cell line ab266971 (knockout cell lysate ab256888). Wild-type and IP10 knockout A549 cell lysates were subjected to SDS-PAGE. Membranes were blocked in fluorescent western blot (TBS-based) blocking solution before incubation with ab214668 and ab8245 (Mouse anti-GAPDH antibody [6C5]) overnight at 4°C at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. Blots were incubated with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 800CW) preabsorbed (ab216772) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

All lanes : Anti-IP10 antibody [EPR20764] (ab214668) at 1/1000 dilution

Lane 1 : Untreated THP-1 (human monocytic leukemia cell line) culture supernatant
Lane 2 : THP-1 treated with 200 ng/ml interferon-gamma (IFN-gamma, ab9659) and 50 ng/ml lipopolysaccharides (LPS) for 24 hours, culture supernatant

Lysates/proteins at 15 µl per lane.

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

Developed using the ECL technique.

Predicted band size: 10 kDa
Observed band size: 12 kDa why is the actual band size different from the predicted?


Exposure time: 15 seconds

Blocking/Dilution buffer: 5% NFDM/TBST.

IP10 protein secretion can be induced by IFN-gamma treatment (PMID: 11907072).

ELISA analysis of CXCL10 recombinant protein at 1000 ng/mL with ab214668. An Alkaline Phosphatase-conjugated AffiniPure Goat Anti-Rabbit IgG (H+L) at 1/2500 dilution was used as the secondary antibody.