Anti-IP10 antibody [EPR7850] - BSA and Azide free

• I-ELISA

Lab

Indirect ELISA - Anti-IP10 antibody [EPR7850] - BSA and Azide free (AB248769)

This data was developed using ab137018, the same antibody clone in a different buffer formulation.ELISA analysis of Human IP10 recombinant protein at 1000 ng/mL with ab137018. An Alkaline Phosphatase-conjugated AffiniPure Goat Anti-Rabbit IgG (H+L) at 1/2500 dilution was used as the secondary antibody.

• WB

Lab

Western blot - Anti-IP10 antibody [EPR7850] - BSA and Azide free (AB248769)

False colour image of Western blot : Anti-IP10 antibody [EPR7850] 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, ab137018 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:

Western blot - Anti-IP10 antibody [EPR7850] (<a href='/en-us/products/primary-antibodies/ip10-antibody-epr7850-ab137018'>ab137018</a>) 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

Lab

Western blot - Anti-IP10 antibody [EPR7850] - BSA and Azide free (AB248769)

This data was developed using the same antibody clone in a different buffer formulation (ab137018).

Lanes 1 - 4 : Merged signal (red and green). Green - ab137018 observed at 11 kDa. Red - loading control ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37kDa.

ab137018 was shown to react with IP10 in A549 wild-type cells in western blot with loss of signal observed in IP10 knockout cell line ab266969 (IP10 knockout cell lysate ab256886). A549 wild-type and IP10 knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in fluorescent western blot (TBS-based) blocking solution before incubation with ab137018 and ab8245 (Mouse anti-GAPDH antibody [6C5]) overnight at 4°C at a 1 in 500 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 [EPR7850] (<a href='/en-us/products/primary-antibodies/ip10-antibody-epr7850-ab137018'>ab137018</a>) at 1/500 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) for 32 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

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-ab266969'>ab266969</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>) (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) for 32 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

Unknown

Western blot - Anti-IP10 antibody [EPR7850] - BSA and Azide free (AB248769)

This data was developed using ab137018, the same antibody clone in a different buffer formulation.

All lanes:

Western blot - Anti-IP10 antibody [EPR7850] (<a href='/en-us/products/primary-antibodies/ip10-antibody-epr7850-ab137018'>ab137018</a>) at 1/1000 dilution

All lanes:

IP10 recombinant protein at 0.01 µg

Secondary

All lanes:

HRP labelled goat anti-rabbit at 1/2000 dilution

Predicted band size: 10 kDa

false