Anti-IP10 antibody [EPR20764]

• WB

Lab

Western blot - Anti-IP10 antibody [EPR20764] (AB214668)

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:

Western blot - Anti-IP10 antibody [EPR20764] (ab214668) at 1/1000 dilution

Lane 1:

Wild-type A549 Brefeldin A (<a href='/en-us/products/biochemicals/brefeldin-a-inhibitor-of-adp-ribosylation-factor-ab120299'>ab120299</a>)-treated (5ug/ml, 6h) cell lysate at 30 µg

Lane 2:

Wild-type A549 IFN-y (<a href='/en-us/products/proteins-peptides/recombinant-human-interferon-gamma-protein-active-ab259377'>ab259377</a>) (100 ng/ml, 32 h) and TNF-alpha (<a href='/en-us/products/proteins-peptides/recombinant-human-tnf-alpha-protein-active-ab259410'>ab259410</a>) (10 ng/ml, 32h), and Brefeldin A (<a href='/en-us/products/biochemicals/brefeldin-a-inhibitor-of-adp-ribosylation-factor-ab120299'>ab120299</a>)-treated (5ug/ml for the last 6h) cell lysate at 30 µg

Lane 2:

Western blot - Human CXCL10 (IP10) knockout THP-1 cell line (<a href='/en-us/products/cell-lines/human-cxcl10-ip10-knockout-thp-1-cell-line-ab277860'>ab277860</a>)

Lane 3:

IP10 knockout A549 Brefeldin A (<a href='/en-us/products/biochemicals/brefeldin-a-inhibitor-of-adp-ribosylation-factor-ab120299'>ab120299</a>)-treated (5ug/ml, 6h) cell lysate at 30 µg

Lane 4:

IP10 knockout A549 IFN-y (<a href='/en-us/products/proteins-peptides/recombinant-human-interferon-gamma-protein-active-ab259377'>ab259377</a>) (100ng/ml, 32h) and TNF-alpha (<a href='/en-us/products/proteins-peptides/recombinant-human-tnf-alpha-protein-active-ab259410'>ab259410</a>) (10ng/ml, 32h), and Brefeldin A (<a href='/en-us/products/biochemicals/brefeldin-a-inhibitor-of-adp-ribosylation-factor-ab120299'>ab120299</a>)-treated (5ug/ml for the last 6h) cell lysate at 30 µg

Lane 5:

THP-1 Brefeldin A (<a href='/en-us/products/biochemicals/brefeldin-a-inhibitor-of-adp-ribosylation-factor-ab120299'>ab120299</a>)-treated (5ug/ml, 6h) cell lysate at 30 µg

Lane 6:

THP-1 IFN-y (<a href='/en-us/products/proteins-peptides/recombinant-human-interferon-gamma-protein-active-ab259377'>ab259377</a>) (200ng/ml, 24h) and LPS (50ng/ml, 24h)-treated for 24 hours, and Brefeldin A (<a href='/en-us/products/biochemicals/brefeldin-a-inhibitor-of-adp-ribosylation-factor-ab120299'>ab120299</a>)-treated (5ug/ml for the last 6h) cell lysate at 30 µg

Predicted band size: 10 kDa

false

• WB

Lab

Western blot - Anti-IP10 antibody [EPR20764] (AB214668)

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:

Western blot - Anti-IP10 antibody [EPR20764] (ab214668) at 1/1000 dilution

Lane 1:

Wild-type A549 Brefeldin A (<a href='/en-us/products/biochemicals/brefeldin-a-inhibitor-of-adp-ribosylation-factor-ab120299'>ab120299</a>)-treated (5ug/ml, 6h) cell lysate at 30 µg

Lane 2:

Wild-type A549 IFN-y (<a href='/en-us/products/proteins-peptides/recombinant-human-interferon-gamma-protein-active-ab259377'>ab259377</a>) (100 ng/ml, 32 h) and TNF-alpha (<a href='/en-us/products/proteins-peptides/recombinant-human-tnf-alpha-protein-active-ab259410'>ab259410</a>) (10 ng/ml, 32h), and Brefeldin A (<a href='/en-us/products/biochemicals/brefeldin-a-inhibitor-of-adp-ribosylation-factor-ab120299'>ab120299</a>)-treated (5ug/ml for the last 6h) cell lysate at 30 µg

Lane 2:

Western blot - Human CXCL10 (IP10) knockout A549 cell line (<a href='/en-us/products/cell-lines/human-cxcl10-ip10-knockout-a549-cell-line-ab266971'>ab266971</a>)

Lane 3:

IP10 knockout A549 Brefeldin A (<a href='/en-us/products/biochemicals/brefeldin-a-inhibitor-of-adp-ribosylation-factor-ab120299'>ab120299</a>)-treated (5ug/ml, 6h) cell lysate at 30 µg

Lane 4:

IP10 knockout A549 IFN-y (<a href='/en-us/products/proteins-peptides/recombinant-human-interferon-gamma-protein-active-ab259377'>ab259377</a>) (100ng/ml, 32h) and TNF-alpha (<a href='/en-us/products/proteins-peptides/recombinant-human-tnf-alpha-protein-active-ab259410'>ab259410</a>) (10ng/ml, 32h), and Brefeldin A (<a href='/en-us/products/biochemicals/brefeldin-a-inhibitor-of-adp-ribosylation-factor-ab120299'>ab120299</a>)-treated (5ug/ml for the last 6h) cell lysate at 30 µg

Lane 5:

THP-1 Brefeldin A (<a href='/en-us/products/biochemicals/brefeldin-a-inhibitor-of-adp-ribosylation-factor-ab120299'>ab120299</a>)-treated (5ug/ml, 6h) cell lysate at 30 µg

Lane 6:

THP-1 IFN-y (<a href='/en-us/products/proteins-peptides/recombinant-human-interferon-gamma-protein-active-ab259377'>ab259377</a>) (200ng/ml, 24h) and LPS (50ng/ml, 24h)-treated for 24 hours, and Brefeldin A (<a href='/en-us/products/biochemicals/brefeldin-a-inhibitor-of-adp-ribosylation-factor-ab120299'>ab120299</a>)-treated (5ug/ml for the last 6h) cell lysate at 30 µg

Predicted band size: 10 kDa

Observed band size: 11 kDa

false

• WB

Lab

Western blot - Anti-IP10 antibody [EPR20764] (AB214668)

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 wild-type A549 cell lysates with no signal observed at this size in CXCL10 knockout cell line ab266970 (knockout cell lysate ab256887). To generate this image, wild-type and CXCL10 knockout A549 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:

Western blot - Anti-IP10 antibody [EPR20764] (ab214668) at 1/1000 dilution

Lane 1:

Wild-type A549 Vehicle Control IFN-gamma (0 ng/mL, 32 h) + TNF-alpha (0 ng/mL, 32h) + BFA (0 u/mL, 6h) cell lysate at 20 µg

Lane 2:

Wild-type A549 Treated IFN-gamma (100 ng/mL, 32 h) + TNF-alpha (10 ng/mL, 32h) + BFA (5 u/mL, 6h) cell lysate at 20 µg

Lane 3:

CXCL10 knockout A549 Vehicle Control IFN-gamma (0 ng/mL, 32 h) + TNF-alpha (0 ng/mL, 32h) + BFA (0 u/mL, 6h) cell lysate at 20 µg

Lane 4:

CXCL10 knockout A549 Treated IFN-gamma (100 ng/mL, 32 h) + TNF-alpha (10 ng/mL, 32h) + BFA (5 u/mL, 6h) cell lysate at 20 µg

Predicted band size: 10 kDa

Observed band size: 11 kDa

false

• WB

Lab

Western blot - Anti-IP10 antibody [EPR20764] (AB214668)

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 ab283141). 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:

Western blot - 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 at 20 µg

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 at 20 µg

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 at 20 µg

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 at 20 µg

Predicted band size: 10 kDa

Observed band size: 11 kDa

false

• WB

Supplier Data

Western blot - Anti-IP10 antibody [EPR20764] (AB214668)

Blocking/Dilution buffer : 5% NFDM/TBST.

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

All lanes:

Western blot - Anti-IP10 antibody [EPR20764] (ab214668) at 1/1000 dilution

Lane 1:

Untreated THP-1 (human monocytic leukemia cell line) culture supernatant at 15 µL

Lane 2:

THP-1 treated with 200 ng/ml interferon-gamma (IFN-gamma, <a href='/en-us/products/proteins-peptides/recombinant-human-interferon-gamma-protein-active-ab9659'>ab9659</a>) and 50 ng/ml lipopolysaccharides (LPS) for 24 hours, culture supernatant at 15 µL

Secondary

All lanes:

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 1/100000 dilution

Predicted band size: 10 kDa

Observed band size: 12 kDa

true

Exposure time: 15s

• ELISA

Unknown

ELISA - Anti-IP10 antibody [EPR20764] (AB214668)

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.