Anti-CXCL5 antibody [EPR4450(2)]

• IP

Lab

Immunoprecipitation - Anti-CXCL5 antibody [EPR4450(2)] (AB126763)

CXCL5 was immunoprecipitated from 0.35 mg A549 (Human lung carcinoma epithelial cell) whole cell lysate 10 μg with 126763 at 1/50 dilution (2μg). VeriBlot for IP Detection Reagent (HRP) (ab131366) was used at 1/5000 dilution.

Lane 1 : A549 (Human lung carcinoma epithelial cell) whole cell lysate 10 μg

Lane 2 : ab126763 IP in A549 whole cell lysate

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab126763 in A549 whole cell lysate

Blocking and dilution buffer and concentration : 5% NFDM/TBST.

All lanes:

Immunoprecipitation - Anti-CXCL5 antibody [EPR4450(2)] (ab126763)

Predicted band size: 12 kDa

Observed band size: 12 kDa

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• WB

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Western blot - Anti-CXCL5 antibody [EPR4450(2)] (AB126763)

All lanes:

Western blot - Anti-CXCL5 antibody [EPR4450(2)] (ab126763) at 1/1000 dilution

Lane 1:

A549 cell lysate, treated with TNF alpha and TPA at 10 µg

Lane 2:

A549 cell lysate, untreated at 10 µg

Lane 3:

Human CXCL5 recombinant protein at 0.001 µg

Secondary

All lanes:

Goat anti-Rabbit HRP at 1/2000 dilution

Predicted band size: 12 kDa

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• WB

Supplier Data

Western blot - Anti-CXCL5 antibody [EPR4450(2)] (AB126763)

False colour image of Western blot : Anti-CXCL5 antibody [EPR4450(2)] staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab126763 was shown to bind specifically to CXCL5. A band was observed at 7 kDa in treated wild-type A549 cell lysates with no signal observed at this size in CXCL5 knockout cell line ab275838 (knockout cell lysate ab275812). To generate this image, wild-type and CXCL5 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 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.

All lanes:

Western blot - Anti-CXCL5 antibody [EPR4450(2)] (ab126763) at 1/1000 dilution

Lane 1:

Wild-type A549 Untreated Control cell lysate at 20 µg

Lane 2:

Wild-type A549 Treated TNFa (10 ng/mL, 24 h) + PMA (10 nM, 24 h) cell lysate at 20 µg

Lane 3:

CXCL5 knockout A549 Untreated Control cell lysate at 20 µg

Lane 4:

CXCL5 knockout A549 Treated TNFa (10 ng/mL, 24 h) + PMA (10 nM, 24 h) cell lysate at 20 µg

Secondary

All lanes:

Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution

Predicted band size: 12 kDa

Observed band size: 7 kDa

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• OI-RD Scanning

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OI-RD Scanning - Anti-CXCL5 antibody [EPR4450(2)] (AB126763)

We have systematically measured KD (the equilibrium dissociation constant between the antibody and its antigen), of more than 840 recombinant antibodies to assess not only their individual KD values but also to see the average affinity of antibody. Based on the comparison with published literature values for mouse monoclonal antibodies, Recombinant antibodies appear to be on average 1-2 order of magnitude higher affinity.