Anti-IP10 antibody [EPR24674-12] - BSA and Azide free

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-IP10 antibody [EPR24674-12] - BSA and Azide free (AB283708)

This data was developed using ab283681, the same antibody clone in a different buffer formulation.

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.

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-IP10 antibody [EPR24674-12] - BSA and Azide free (AB283708)

This data was developed using ab283681, the same antibody clone in a different buffer formulation.

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.

• WB

Lab

Western blot - Anti-IP10 antibody [EPR24674-12] - BSA and Azide free (AB283708)

This data was developed using ab283681, the same antibody clone in a different buffer formulation.

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.

All lanes:

Western blot - Anti-IP10 antibody [EPR24674-12] (<a href='/en-us/products/primary-antibodies/ip10-antibody-epr24674-12-ab283681'>ab283681</a>) 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 (<a href='/en-us/products/proteins-peptides/recombinant-human-interferon-gamma-protein-active-ab259377'>ab259377</a>) for 32 hours and 10 ng/m TNF-alpha (<a href='/en-us/products/proteins-peptides/recombinant-human-tnf-alpha-protein-active-ab259410'>ab259410</a>) for 32 hours, and 5ug/ml Brefeldin A (<a href='/en-us/products/biochemicals/brefeldin-a-inhibitor-of-adp-ribosylation-factor-ab120299'>ab120299</a>) for the last 6 hours, whole cell lysate at 40 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:

Untreated IP10 knockout A549 whole cell lysate at 40 µg

Lane 4:

IP10 knockout A549 treated with 100 ng/ml IFN-y (<a href='/en-us/products/proteins-peptides/recombinant-human-interferon-gamma-protein-active-ab259377'>ab259377</a>) for 32 hours and 10 ng/m TNF-alpha (<a href='/en-us/products/proteins-peptides/recombinant-human-tnf-alpha-protein-active-ab259410'>ab259410</a>) for 32 hours, and 5ug/ml Brefeldin A (<a href='/en-us/products/biochemicals/brefeldin-a-inhibitor-of-adp-ribosylation-factor-ab120299'>ab120299</a>) 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 (<a href='/en-us/products/proteins-peptides/recombinant-human-interferon-gamma-protein-active-ab259377'>ab259377</a>) 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) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-800cw-preadsorbed-ab216773'>ab216773</a>) and Goat Anti-Mouse IgG H&L (IRDye® 680RD) (<a href='/en-us/products/secondary-antibodies/goat-mouse-igg-h-l-irdye-680rd-preadsorbed-ab216776'>ab216776</a>) at 1/10000 dilution

Predicted band size: 10 kDa

Observed band size: 11 kDa

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