Anti-TNF alpha antibody [RM1005]

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-TNF alpha antibody [RM1005] (AB307164)

Flow cytometric analysis of 2% paraformaldehyde fixed 0.1% Tween-20 permeabilized Human peripheral blood mononuclear cell (PBMC) treated with 50ng/ml PMA, 200ng/ml Ionomycin calcium and 300ng/ml BFA for 4h (Right) / Untreated control (Left) cells labelling TNF alpha with ab307164 at 1/500 dilution (0.1ug)/ Left and Right (Red) compared with a 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. Cells were surface stained with anti-CD3 conjugated to Alexa Fluor^® 647. Then fixed with 2% PFA for 10min followed by intracellularly stained with ab307164.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-TNF alpha antibody [RM1005] (AB307164)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized Human PBMC (human peripheral blood mononuclear cell) cells labeling TNF alpha with ab307164 at 1/500 (1.12 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 human PBMC treated with Phorbol-12-myristate-13-acetate (50ng/ml) and Ionomycin calcium (200ng/ml) and Brefeldin A (300ng/ml) for 4 hours. 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 dilution (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.

• IP

Supplier Data

Immunoprecipitation - Anti-TNF alpha antibody [RM1005] (AB307164)

TNF alpha was immunoprecipitated from 0.35 mg THP-1 (human monocytic leukemia monocyte) treated with 80nM TPA overnight, then treated with 100ng/ml LPS for 3h and add 300ng/ml BFA for another 3h whole cell lysate 5 ug with ab307164 at 1/30 dilution (2ug in 0.18mg lysates). Western blot was performed on the immunoprecipitate using ab307164 at 1/1000 dilution. VeriBlot for IP secondary antibody (HRP) (ab131366) was used at 1/5000 dilution.

Lane 1 : THP-1 (human monocytic leukemia monocyte) treated with 80nM TPA overnight, then treated with 100ng/ml LPS for 3h and add 300ng/ml BFA for another 3h whole cell lysate 5 ug

Lane 2 : ab307164 IP in THP-1 treated with 80nM TPA overnight, then treated with 100ng/ml LPS for 3h and add 300ng/ml BFA for another 3h whole cell lysate

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab307164 in THP-1 treated with 80nM TPA overnight, then treated with 100ng/ml LPS for 3h and add 300ng/ml BFA for another 3h whole cell lysate

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

Exposure time : 6 seconds

Soluble form (17 kDa) and multimers of soluble form (33 kDa, 51 kDa) were also observed as described in literatures (PMID : 18523283, PMID : 28426652).

All lanes:

Immunoprecipitation - Anti-TNF alpha antibody [RM1005] (ab307164) at 1/1000 dilution

Lane 1:

THP-1 (human monocytic leukemia monocyte) treated with 80nM TPA overnight, then treated with 100ng/ml LPS for 3h and add 300ng/ml BFA for another 3h whole cell lysate 5 μg

Lane 2:

THP-1 treated with 80nM TPA overnight, then treated with 100ng/ml LPS for 3h and add 300ng/ml BFA for another 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

Observed band size: 17 kDa,26 kDa,51 kDa

false

Exposure time: 6s

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-TNF alpha antibody [RM1005] (AB307164)

Immunohistochemical analysis of paraffin-embedded Mouse lung treated w tissue labeling TNF alpha with ab307164 at 1/1000 (0.56 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Positive staining on mouse lung treated with LPS (1ug/ml for 16 hours) and BFA (1ug/ml for 16h hours) (Image A) and control mouse lung (Image B). The section was incubated with ab307164 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND™ RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-TNF alpha antibody [RM1005] (AB307164)

Immunohistochemical analysis of paraffin-embedded Mouse cerebrum tissue labeling TNF alpha with ab307164 at 1/1000 (0.56 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Negative control : no staining on mouse cerebrum. The section was incubated with ab307164 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND™ RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-TNF alpha antibody [RM1005] (AB307164)

Immunohistochemical analysis of formalin fixed paraffin embedded rat lung labelling TNF alpha with ab307164 at a concentration of 1/1000 dilution. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins. The primary antibody Anti-TNF alpha antibody [RM1005] was incubated for 30 mins at room temperature. Sections were counterstained with Hematoxylin.

Positive staining on rat lung treated with LPS (1ug/ml for 16 hours) and BFA (1ug/ml for 16h hours) (Image A) and no staining on wild type rat lung (Image B).

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-TNF alpha antibody [RM1005] (AB307164)

Immunohistochemical analysis of formalin fixed paraffin embedded rat cerebrum labelling TNF alpha with ab307164 at a concentration of 1/1000 dilution. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins. The primary antibody Anti-TNF alpha antibody [RM1005] was incubated for 30 mins at room temperature. Sections were counterstained with Hematoxylin.

Negative control : no staining on rat cerebrum.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-TNF alpha antibody [RM1005] (AB307164)

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 and add 1µg/ml BFA for another 3h (Red) / Untreated control (Green) cells labelling TNF alpha with ab307164 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.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-TNF alpha antibody [RM1005] (AB307164)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized RAW 264.7 (mouse abelson murine leukemia virus-induced tumor macrophage) cells labeling TNF alpha with ab307164 at 1/500 (1.12 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 RAW 264.7 treated with lipopolysaccharide (100 ng/ml) and Brefeldin A (1 µg/ml) for 3 hours. 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 dilution (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.

• IP

Supplier Data

Immunoprecipitation - Anti-TNF alpha antibody [RM1005] (AB307164)

TNF alpha was immunoprecipitated from 0.35 mg RAW 264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage) treated with 100ng/ml LPS for 4h and add 300ng/ml BFA for another 3h whole cell lysate 10 ug with ab307164 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab307164 at 1/1000 dilution. VeriBlot for IP secondary antibody (HRP) (ab131366) was used at 1/5000 dilution.

Lane 1 : RAW 264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage) treated with 100ng/ml LPS for 4h and add 300ng/ml BFA for another 3h whole cell lysate 10 ug

Lane 2 : ab307164 IP in RAW 264.7 treated with 100ng/ml LPS for 4h and add 300ng/ml BFA for another 3h whole cell lysate

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab307164 in RAW 264.7 treated with 100ng/ml LPS for 4h and add 300ng/ml BFA for another 3h whole cell lysate

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

Exposure time : 6 seconds

Soluble form (17 kDa) and multimers of soluble form (33 kDa, 51 kDa) were also observed as described in literatures (PMID : 18523283, PMID : 28426652).

All lanes:

Immunoprecipitation - Anti-TNF alpha antibody [RM1005] (ab307164) at 1/1000 dilution

Lane 1:

RAW 264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage) treated with 100ng/ml LPS for 4h and add 300ng/ml BFA for another 3h whole cell lysate 10 μg

Lane 2:

RAW 264.7 treated with 100ng/ml LPS for 4h and add 300ng/ml BFA for another 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

Observed band size: 17 kDa,26 kDa,33 kDa,51 kDa

false

Exposure time: 6s

• WB

Supplier Data

Western blot - Anti-TNF alpha antibody [RM1005] (AB307164)

Blocking and diluting buffer and concentration : 5% NFDM/TBST Soluble form (17 kDa) and multimers of soluble form (33 kDa, 51 kDa) were also observed as described in literatures (PMID : 18523283, PMID : 28426652). Exposure time : 180 seconds

All lanes:

Western blot - Anti-TNF alpha antibody [RM1005] (ab307164) at 1/1000 dilution

Lane 1:

THP-1 (human monocytic leukemia monocyte) whole cell lysate 20 μg

Lane 2:

THP-1 treated with 80nM TPA overnight, then treated with /ml LPS for 3h and add 300ng/ml BFA for another 3h whole cell lysate 20 μg

Lane 3:

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

Lane 4:

RAW 264.7 treated with /ml LPS for 4h and add 300ng/ml BFA for another 3h whole cell lysate 20 μg

Lane 5:

NR8383 (rat alveolar macrophage) whole cell lysate 20 μg

Lane 6:

NR8383 treated with /ml LPS for 4h and add 300ng/ml BFA for another 3h whole cell lysate 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: 17 kDa,26 kDa,33 kDa,51 kDa

false

Exposure time: 180s

• I-ELISA

Supplier Data

Indirect ELISA - Anti-TNF alpha antibody [RM1005] (AB307164)

Indirect ELISA analysis of ab173305 at 1/1000 ng/ml. The secondary antibody used was Alkaline Phosphatase-conjugated AffiniPure Goat Anti-Rabbit IgG (H+L) at 1/2500 dilution. Substrate solution : p-nitrophenyl phosphate(PNPP).