Anti-IL-32 antibody [EPR28310-70]

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-IL-32 antibody [EPR28310-70] (AB315466)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized A549 (human lung carcinoma epithelial cell) cells labelling IL-32 with ab315466 at 1/50 (10.38 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 TNFα (25 ng/mL) for 24 hr.
Image was taken with a confocal microscope(Leica-Microsystems, TCS SP8).

ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 (2.5ug/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.

• IP

Supplier Data

Immunoprecipitation - Anti-IL-32 antibody [EPR28310-70] (AB315466)

IL-32 was immunoprecipitated from 0.35 mg A549 (human lung carcinoma epithelial cell) treated with 25 ng/ml TNF alpha for 24 hours, whole cell lysate with ab315466 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab315466 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

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

All lanes:

Immunoprecipitation - Anti-IL-32 antibody [EPR28310-70] (ab315466) at 1/1000 dilution

Lane 1:

A549 (human lung carcinoma epithelial cell) treated with 25 ng/ml TNF alpha for 24 hours, whole cell lysate

Lane 2:

ab315466 at 1/30 IP in A549 (human lung carcinoma epithelial cell) treated with 25 ng/ml TNF alpha for 24 hours, whole cell lysate

Lane 3:

Rabbit monoclonal IgG (<a href='/en-us/products/primary-antibodies/rabbit-igg-monoclonal-epr25a-isotype-control-ab172730'>ab172730</a>) instead of ab315466 in A549 treated with 25 ng/ml TNF alpha for 24h, 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

false

Exposure time: 8s

• WB

Supplier Data

Western blot - Anti-IL-32 antibody [EPR28310-70] (AB315466)

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

The expression of IL-32 is upregulated in response to TNF alpha treatment (PMID : 24633341, 30115930).

The identity of the higher MW band at approximately 70kDa is unknown.

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/200000 dilution.

All lanes:

Western blot - Anti-IL-32 antibody [EPR28310-70] (ab315466) at 1/1000 dilution

Lane 1:

Untreated A549 (human lung carcinoma epithelial cell) whole cell lysate at 20 µg

Lane 2:

A549 treated with 25 ng/ml TNF alpha for 24 hours, 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: 25 kDa,36 kDa

false

Exposure time: 180s

• WB

Supplier Data

Western blot - Anti-IL-32 antibody [EPR28310-70] (AB315466)

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

Negative control : A549, PC-3, Daudi.

The expression profile/ molecular weight observed is consistent with what has been described in the literature (PMID : 16410314).

The identity of the higher MW band at approximately 70kDa is unknown.

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/200000 dilution.

All lanes:

Western blot - Anti-IL-32 antibody [EPR28310-70] (ab315466) at 1/1000 dilution

Lane 1:

No-GFP-CD16.NK-92 (CD16 ectopically expressed natural killer(NK-92®) cell with no GFP tag) whole cell lysate at 20 µg

Lane 2:

A549 (human lung carcinoma epithelial cell) whole cell lysate at 20 µg

Lane 3:

PC-3 (human prostate adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 4:

Daudi (human Burkitts lymphoma lymphoblast) 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: 25 kDa,20 kDa,36 kDa

false

Exposure time: 3.25s