Anti-IL-6- antibody [EPRNGS_IL-6-20] BSA and Azide free, low endotoxin

• FuncS (Neut/Block)

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Functional Studies (Neut/Block) - Anti-IL-6- antibody [EPRNGS_IL-6-20] BSA and Azide free, low endotoxin (AB323372)

Cell proliferation of TF-1 (human erythroleukemia erythroblast) cells treated with IL-6 (ab259381) and anti-IL-6 antibody (ab323372) or isotype IgG (ab210849).​

Cell proliferation (%) (A) confirms that the anti-IL-6 antibody (ab323372) neutralizes IL-6-mediated cell proliferation. The IC50 as determined by the dose-dependent inhibition of TF-1 cells proliferation is 4.990 ng/ml (B). ​

TF-1 cells were seeded at 5 × 10⁴ cells per well in a 96-well plate and cultured in medium containing 5% FBS for 48 h. Proliferation was analysed by luminescent assay and recorded using a Microplate Reader at 560nm emission.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-IL-6- antibody [EPRNGS_IL-6-20] BSA and Azide free, low endotoxin (AB323372)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized HUVEC (human umbilical vein endothelial cell) treated with 0.5ug/mL LPS for 24 hours and 300ng/mL BFA for 20 hours (Green) / Untreated HUVEC (Magenta) cells labeling IL-6 with ab323372 at 1/1000 dilution (0.1 ug) / Magenta and Green compared with a Rabbit monoclonal IgG (ab172730) (Black) and (Grey) isotype control and an unlabeled control (cells without incubation with primary antibody and secondary antibody).

Goat Anti-Rabbit IgG (Alexa Fluor® 488, ab150081) at 1/2000 dilution was used as the secondary antibody.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-IL-6- antibody [EPRNGS_IL-6-20] BSA and Azide free, low endotoxin (AB323372)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized HUVEC (human umbilical vein endothelial cell) cells labelling IL-6 with ab323372 at 1/100 (10 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 2ug/ml dilution (Green).

Confocal image showing increased cytoplasmic staining in HUVEC cells treated with lipopolysaccharide (0.5 ug/ml) for 24 h, then Brefeldin A (300ng/ml) for another 20 h (shown in green). The counterstain was observed in magenta. Nuclear DNA was labelled with DAPI (shown in blue). 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 (Magenta). 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 2ug/ml dilution.

• IP

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Immunoprecipitation - Anti-IL-6- antibody [EPRNGS_IL-6-20] BSA and Azide free, low endotoxin (AB323372)

IL-6 was immunoprecipitated from 0.35 mg HUVEC (human umbilical vein endothelial cell) treated with 0.5µg/ml LPS for 24h, 300ng/ml BFA was then added for additional 20h, whole cell lysate with ab323372 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab323372 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1 : HUVEC (human umbilical vein endothelial cell) treated with 0.5µg/ml LPS for 24h, 300ng/ml BFA was then added for additional 20h, whole cell lysate
Lane 2 : ab323372 IP in HUVEC (human umbilical vein endothelial cell) treated with 0.5µg/ml LPS for 24h, 300ng/ml BFA was then added for additional 20h, whole cell lysate
Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab323372 in HUVEC treated with 0.5µg/ml LPS for 24h, 300ng/ml BFA was then added for additional 20h, whole cell lysate

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

All lanes:

Immunoprecipitation - Anti-IL-6- antibody [EPRNGS_IL-6-20] BSA and Azide free, low endotoxin (ab323372) at 1/30 dilution

All lanes:

HUVEC (human umbilical vein endothelial cell) treated with 0.5µg/ml LPS for 24h, 300ng/ml BFA was then added for additional 20h, whole cell lysate

Secondary

All lanes:

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

false

Exposure time: 24s

• WB

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Western blot - Anti-IL-6- antibody [EPRNGS_IL-6-20] BSA and Azide free, low endotoxin (AB323372)

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

The expression of IL-6 is upregulated in response to LPS and BFA treatment.

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

All lanes:

Western blot - Anti-IL-6- antibody [EPRNGS_IL-6-20] BSA and Azide free, low endotoxin (ab323372) at 1/1000 dilution

Lane 1:

Untreated HUVEC (human umbilical vein endothelial cell) whole cell lysate at 20 µg

Lane 2:

HUVEC (human umbilical vein endothelial cell) treated with 0.5 ug/ml LPS for 24h, 300ng/ml BFA was then added for additional 20h, whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/ko/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Observed band size: 23-28 kDa,36 kDa

false

Exposure time: 180s

• I-ELISA

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Indirect ELISA - Anti-IL-6- antibody [EPRNGS_IL-6-20] BSA and Azide free, low endotoxin (AB323372)

Indirect ELISA analysis of ab323372 at 1000-0 ng/ml. The Secondary antibody used was Alkaline Phosphatase-conjugated AffiniPure Goat Anti-Rabbit IgG (H+L) at 1 : 2500 dilution dilution.

Antigen : Human IL-6 protein.

Antigen concentration : 1000 ng/ml