Anti-CXCL5 antibody [EPR23928-217]

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-CXCL5 antibody [EPR23928-217] (AB305100)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized A549 (human lung carcinoma epithelial cell) cells labelling CXCL5 with ab305100 at 1/500 (1.012 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 increased cytoplasmic staining in A549 cells treated with starved overnight, then add TPA (10 nM), TNF alpha (10 ng/ml) and BSA (0.1%) for 24 h and treated with BFA (300 ng/ml) for another 3 h. The image was taken with a confocal microscope (Leica-Microsystems, TCS SP8). is observed. 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.

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-CXCL5 antibody [EPR23928-217] (AB305100)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized A549 (human lung carcinoma epithelial cell) treated with 10ng/ml TNF alpha and 10nM TPA and 0.1% BSA for 24 hours, then add 300ng/ml BFA for another 3 hours (Red) / Untreated control (Green) cells labelling CXCL5 with ab305100 at 1/500 dilution (0.1ug) (Red) and Green (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat Anti-Rabbit IgG (Alexa Fluor® 488, ab150081) at 1/2000 dilution was used as the secondary antibody.

• IP

Supplier Data

Immunoprecipitation - Anti-CXCL5 antibody [EPR23928-217] (AB305100)

CXCL5 was immunoprecipitated from 0.35 mg A549 (human lung carcinoma epithelial cell) starved overnight, then treated with 10 ng/ml TNF alpha, 10 nM TPA (ab120297), and 0.1% BSA for 24h, then with 300 ng/ml BFA added for 3h, whole cell lysate 10 µg with ab305100 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab305100 at 1/1000 dilution. VeriBlot for IP secondary antibody (HRP) (ab131366) was used at 1/5000 dilution.

Lane 1 : A549 (human lung carcinoma epithelial cell) starved overnight, then treated with 10 ng/ml TNF alpha, 10 nM TPA (ab120297), and 0.1% BSA for 24h, then with 300 ng/ml BFA added for 3h, whole cell lysate 10 µg

Lane 2 : ab305100 IP in A549 starved overnight, then treated with 10 ng/ml TNF alpha, 10 nM TPA (ab120297), and 0.1% BSA for 24h, then with 300 ng/ml BFA added for 3h, whole cell lysate

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab305069 in A549 starved overnight, then treated with 10 ng/ml TNF alpha, 10 nM TPA (ab120297), and 0.1% BSA for 24h, then with 300 ng/ml BFA added for 3h, whole cell lysate

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

Exposure time : 3 minutes.

All lanes:

Immunoprecipitation - Anti-CXCL5 antibody [EPR23928-217] (ab305100) at 1/1000 dilution

Lane 1:

A549 (human lung carcinoma epithelial cell) starved overnight, then treated with 10 ng/ml TNF alpha, 10 nM TPA (<a href='/en-us/products/biochemicals/phorbol-12-myristate-13-acetate-pma-pkc-activator-ab120297'>ab120297</a>), and 0.1% BSA for 24h, then with 300 ng/ml BFA added for 3h, whole

Lane 2:

ab305100 IP in A549 starved overnight, then treated with 10 ng/ml TNF alpha, 10 nM TPA (<a href='/en-us/products/biochemicals/phorbol-12-myristate-13-acetate-pma-pkc-activator-ab120297'>ab120297</a>), and 0.1% BSA for 24h, then with 300 ng/ml BFA added for 3h, whole cell lysate

Secondary

All lanes:

Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/5000 dilution

Predicted band size: 12 kDa

Observed band size: 12 kDa

false

Exposure time: 3min

• WB

Supplier Data

Western blot - Anti-CXCL5 antibody [EPR23928-217] (AB305100)

Blocking and diluting buffer and concentration : 5% NFDM/TBST This antibody does not cross-react with human CXCL6. Exposure time : 37 seconds

All lanes:

Western blot - Anti-CXCL5 antibody [EPR23928-217] (ab305100) at 1/1000 dilution

Lane 1:

HEK-293T cells transfected with a human CXCL5 expression vector containing a myc-his tag, then treated with 600 /ml BFA (<a href='/en-us/products/biochemicals/brefeldin-a-inhibitor-of-adp-ribosylation-factor-ab120299'>ab120299</a>) for 3h, whole cell lysate at 20 µg

Lane 2:

HEK-293T cells transfected with a human CXCL6 expression vector containing a myc-his tag, then treated with 600 /ml BFA (<a href='/en-us/products/biochemicals/brefeldin-a-inhibitor-of-adp-ribosylation-factor-ab120299'>ab120299</a>) for 3h, whole cell lysate at 20 µg

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

Observed band size: 16 kDa

false

Exposure time: 37s

• WB

Supplier Data

Western blot - Anti-CXCL5 antibody [EPR23928-217] (AB305100)

Blocking and diluting buffer and concentration : 5% NFDM/TBST The expression of CXCL5 is upregulated in response to TNF-a and PMA treatment (PMID : 9057843; PMID : 23922745). The identity of the higher MW band at approximately 70 kDa is unknown. Exposure time : 125 seconds

All lanes:

Western blot - Anti-CXCL5 antibody [EPR23928-217] (ab305100) at 1/1000 dilution

Lane 1:

A549 (human lu carcinoma epithelial cell) starved overnight, whole cell lysate at 20 µg

Lane 2:

A549 starved overnight, then treated with /ml TNF alpha, 10 nM TPA (<a href='/en-us/products/biochemicals/phorbol-12-myristate-13-acetate-pma-pkc-activator-ab120297'>ab120297</a>), and 0.1% BSA for 24h, then with 300 ng/ml BFA added for 3h, whole cell lysate at 20 µg

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 1/20000 dilution

Observed band size: 12 kDa

false

Exposure time: 125s