Anti-TNF alpha antibody [EPR21753-109] - BSA and Azide free

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-TNF alpha antibody [EPR21753-109] - BSA and Azide free (AB252382)

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized RAW 264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage) (+/- treatment with 100ng/ml LPS for 3h, then together with 300ng/ml BFA for another 3h) cells labeling TNF alpha with ab205587 at 1/100 dilution, followed by a AlexaFluor^®488 Goat anti-Rabbit secondary (ab150077) at 1/1000 dilution (Green). Confocal image showing cytoplasmic staining in RAW 264.7 cells treated with 100ng/ml LPS for 3h, then together with 300ng/ml BFA for another 3h. The Nuclear counterstain is DAPI (Blue). Tubulin was stained using an Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594, ab195889) at 1/200 dilution.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is AlexaFluor^®488 Goat anti-Rabbit secondary (ab150077) at 1/1000 dilution.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab205587).

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-TNF alpha antibody [EPR21753-109] - BSA and Azide free (AB252382)

Intracellular flow cytometric analysis of 4% paraformaldehyde-fixed 90% methanol-permeabilized RAW 264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage) treated with 100ng/ml LPS for 3h, then together with 300ng/ml BFA for another 3h (Red) / Untreated control (Green), compared to an unlabeled control (cells without incubation with primary antibody and secondary antibody, Blue) and an isotype control- Rabbit monoclonal IgG (ab172730, Black). A Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077)at 1/2000 dilution was used as the secondary antibody.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab205587).

• IP

Unknown

Immunoprecipitation - Anti-TNF alpha antibody [EPR21753-109] - BSA and Azide free (AB252382)

TNF alpha was immunoprecipitated from 0.35 mg rat splenocytes (treated with 20ng/ml PMA, 1μg/ml Ionomycin and 10μM BFA for 6h) whole cell lysate with ab205587 at 1/50 dilution. Western blot was performed on the immunoprecipitate using ab205587 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366) was used for detection at 1/5000 dilution.

Lane 1 : Rat splenocytes (treated with 20ng/ml PMA, 1μg/ml Ionomycin and 10μM BFA for 6h) whole cell lysate 10μg (input).

Lane 2 : ab205587 IP in rat splenocytes (treated with 20ng/ml PMA, 1μg/ml Ionomycin and 10μM BFA for 6h) whole cell lysate.

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab205587 in rat splenocytes (treated with 20ng/ml PMA, 1μg/ml Ionomycin and 10μM BFA for 6h) whole cell lysate.

Blocking and dilution buffer and concentration : 5% NFDM/TBST.
Exposure time : 30 seconds.

The MW observed is consistent with what has been described in the literature (PMID : 9933416).

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab205587).

All lanes:

Immunoprecipitation - Anti-TNF alpha antibody [EPR21753-109] (<a href='/en-us/products/primary-antibodies/tnf-alpha-antibody-epr21753-109-ab205587'>ab205587</a>)

Predicted band size: 25 kDa

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• WB

Lab

Western blot - Anti-TNF alpha antibody [EPR21753-109] - BSA and Azide free (AB252382)

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab205587).

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

Normal tissues express undetectable level of TNF alpha, IL-1 beta and IL-6 proteins.

IL-1 beta and IL-6 could be a good control validating the increased TNF alpha level after drug treatment.

All lanes:

Western blot - Anti-TNF alpha antibody [EPR21753-109] (<a href='/en-us/products/primary-antibodies/tnf-alpha-antibody-epr21753-109-ab205587'>ab205587</a>) at 1/1000 dilution

Lane 1:

Rat cerebral cortex tissue lysate at 20 µg

Lane 2:

Rat cerebellum tissue lysate at 20 µg

Lane 3:

Rat hippocampus tissue lysate at 20 µg

Lane 4:

Rat spinal cord tissue lysate at 20 µg

Lane 5:

Rat small intestine tissue lysate at 20 µg

Lane 6:

Rat kidney tissue lysate at 20 µg

Lane 7:

Rat skin tissue lysate at 20 µg

Lane 8:

RAW 264.7 (Mouse Abelson murine leukemia virus-induced tumor macrophage) whole cell lysate at 20 µg

Lane 9:

RAW 264.7 (Mouse Abelson murine leukemia virus-induced tumor macrophage) treated with 100ng/ml lipopolysaccharide (LPS) for 4h then add 1000ng/ml BFA for another 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: 23 kDa,26 Da

false

Exposure time: 180s

• WB

Lab

Western blot - Anti-TNF alpha antibody [EPR21753-109] - BSA and Azide free (AB252382)

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab205587).

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

IL-1 beta and IL-6 could be a good control validating the increased TNF alpha level after drug treatment.

Lanes 1 - 6:

Western blot - Anti-TNF alpha antibody [EPR21753-109] (<a href='/en-us/products/primary-antibodies/tnf-alpha-antibody-epr21753-109-ab205587'>ab205587</a>) at 1/1000 dilution

Lanes 1 - 6:

Western blot - Anti-TNF alpha antibody [EPR21753-109] - BSA and Azide free (ab252382) at 1/1000 dilution

Lanes 1, 3 and 5:

Untreated NR8383 (rat lung macrophage (alveolar)) whole cell lysate at 20 µg

Lane 2:

NR8383 (rat lung macrophage (alveolar)) treated with 100ng/ml lipopolysaccharide (LPS) for 4h then add 1000ng/ml BFA for another 3h whole cell lysate at 20 µg

Lane 4:

NR8383 (rat lung macrophage (alveolar)) treated with 100ng/ml lipopolysaccharide (LPS) for 3h then add 300ng/ml BFA for another 3h whole cell lysate at 20 µg

Lane 6:

NR8383 (rat lung macrophage (alveolar)) treated with 1000ng/ml lipopolysaccharide (LPS) for 2h 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: 23 kDa,26 kDa

false

Exposure time: 20s