Anti-TNF alpha antibody [EPR20972]

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-TNF alpha antibody [EPR20972] (AB215188)

Immunohistochemical analysis of formalin fixed paraffin embedded mouse lung labelling TNF alpha with ab215188 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 [EPR20972] was incubated for 30 mins at room temperature. Sections were counterstained with Hematoxylin. Positive staining on mouse lung treated with LPS (1ug/ml for 16 hours) and BFA (1ug/ml for 16h hours) (Image A) and no staining on wild type mouse lung (Image B).

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-TNF alpha antibody [EPR20972] (AB215188)

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized RAW 264.7 (mouse macrophage cell line transformed with Abelson murine leukemia virus) cells, untreated or treated with 100 ng/ml lipopolysaccharides (LPS) for 7 hours with addition of 1 μg/ml brefeldin A (BFA) for the last 3 hours labeling TNF alpha with ab215188 at 1/100 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Cytoplasmic staining was increased on RAW 264.7 cells when treated with 100 ng/ml lipopolysaccharides (LPS) for 7 hours with addition of 1 μg/ml brefeldin A (BFA) for the last 3 hours.

The nuclear counter stain is DAPI (blue). Tubulin is detected with Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) (ab195889) (red) at 1/200 dilution.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-TNF alpha antibody [EPR20972] (AB215188)

Intracellular flow cytometric analysis of 4% paraformaldehyde-fixed, 0.1% Tween-20 permeabilized mouse splenocytes treated with 20ng/ml PMA, 1μg/ml Ionomycin and 10μM Brefeldin A for 6 hours labeling TNF alphawith ab215188 at 1/600 dilution (right panel) compared with a Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730) (left panel). Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) at 1/2000 dilution was used as the secondary antibody.

Cells were surface stained with anti-mouse CD3, fixed with 4% PFA for 10 minutes, then permeabilized with 0.1% Tween-20 and intracellular stained with anti-rabbit IgG and ab215188. TNF alpha is mainly expressed in T cells (CD3+ population) while only a small population of CD3- cells can express TNF-alpha.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-TNF alpha antibody [EPR20972] (AB215188)

Immunohistochemical analysis of formalin fixed paraffin embedded rat lung labelling TNF alpha with ab215188 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 [EPR20972] 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).

• IP

Supplier Data

Immunoprecipitation - Anti-TNF alpha antibody [EPR20972] (AB215188)

TNF alpha was immunoprecipitated from 0.35 mg of RAW 264.7 (mouse macrophage cell line transformed with Abelson murine leukemia virus) treated with 100 ng/ml lipopolysaccharides (LPS) for 7 hours with addition of 1 μg/ml brefeldin A (BFA) for the last 3 hours, whole cell lysate with ab215188 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab215188 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10,000 dilution

Lane 1 : RAW 264.7 treated with 100 ng/ml lipopolysaccharides (LPS) for 7 hours with addition of 1 μg/ml brefeldin A (BFA) for the last 3 hours, whole cell lysate 10 μg (Input).

Lane 2 : ab215188 IP in RAW 264.7 treated with 100 ng/ml lipopolysaccharides (LPS) for 7 hours with addition of 1 μg/ml brefeldin A (BFA) for the last 3 hours, whole cell lysate (+).

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab215188 in RAW 264.7 treated with 100 ng/ml lipopolysaccharides (LPS) for 7 hours with addition of 1 μg/ml brefeldin A (BFA) for the last 3 hours, whole cell lysate (-).

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

Exposure time : 3 seconds.

All lanes:

Immunoprecipitation - Anti-TNF alpha antibody [EPR20972] (ab215188)

Predicted band size: 25 kDa

Observed band size: 26 kDa,33 kDa

false

• WB

Supplier Data

Western blot - Anti-TNF alpha antibody [EPR20972] (AB215188)

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-TNF alpha antibody [EPR20972] (ab215188) at 1/1000 dilution

Lane 1:

THP-1 (human monocytic leukemia cell line) differentiated with 100 nM TPA overnight, whole cell lysate at 10 µg

Lane 2:

THP-1 (human monocytic leukemia cell line) differentiated with 100 nM TPA overnight, then treated with 100 ng/ml lipopolysaccharides (LPS) for 7 hours with addition of 1 μg/ml brefeldin A (BFA) for the last 3 hours, whole cell lysate at 10 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 1/50000 dilution

Predicted band size: 25 kDa

Observed band size: 26 kDa

true

Exposure time: 3min

• WB

Lab

Western blot - Anti-TNF alpha antibody [EPR20972] (AB215188)

This Western blot image is a comparison between ab215188 and ab183218 tested under the same conditions. While ab215188 is suitable for WB for some samples, ab183218 was found to be more sensitive. False colour image of Western blot : Anti-TNF alpha antibody [EPR20972] staining at 1/1000 dilution, shown in green; Mouse anti-Alpha Tubulin [DM1A] (ab7291) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab215188 was shown to bind specifically to TNF alpha. A band was observed at 27 kDa in treated U937 cell lysates with no signal observed at this size without treatment. No signal was observed in wild-type THP-1 cell lysates or in TNF knockout cell line ab273761 (knockout cell lysate ab275507) with ab215188. However, a band was observed at 27 kDa in treated wild-type THP-1 cell lysates with ab183218. To generate this image, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in fluorescent western blot (TBS-based) blocking solution before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (ab216776) at 1/20000 dilution.

All lanes:

Western blot - Anti-TNF alpha antibody [EPR20972] (ab215188) at 1/1000 dilution

Lane 1:

Wild-type THP-1 control: Brefeldin A (5 ug/mL, 4 h) cell lysate at 30 µg

Lane 2:

Wild-type treated THP-1: LPS (100 ng/mL, 16 h), Brefeldin A (5 ug/mL, last 4 h) cell lysate at 30 µg

Lane 2:

Western blot - Human TNF knockout THP-1 cell line (<a href='/en-us/products/cell-lines/human-tnf-knockout-thp-1-cell-line-ab273761'>ab273761</a>)

Lane 3:

TNF alpha knockout THP-1 control: Brefeldin A (5 ug/mL, 4 h) cell lysate at 30 µg

Lane 4:

TNF alpha knockout THP-1 treated: LPS (100 ng/mL, 16 h), Brefeldin A (5 ug/mL, last 4 h) cell lysate at 30 µg

Lane 5:

U937 control: PMA (10 mM, 2 days), Brefeldin A (5 ug/mL, last 4 h) cell lysate at 30 µg

Lane 6:

U937 treated: PMA (10 mM, 2 days), LPS (1 ug/mL, last 16 h), Brefeldin A (5 ug/mL, last 4 h) cell lysate at 30 µg

Predicted band size: 25 kDa

Observed band size: 27 kDa

false

• WB

Supplier Data

Western blot - Anti-TNF alpha antibody [EPR20972] (AB215188)

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-TNF alpha antibody [EPR20972] (ab215188) at 1/1000 dilution

Lane 1:

Untreated RAW 264.7 (mouse macrophage cell line transformed with Abelson murine leukemia virus), whole cell lysate at 10 µg

Lane 2:

RAW 264.7 (mouse macrophage cell line transformed with Abelson murine leukemia virus), treated with 100 ng/ml lipopolysaccharides (LPS) for 4 hours, 1µg/ml Brefeldin A (BFA) was then added for additional 3 hours, whole cell lysate at 10 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

Predicted band size: 25 kDa

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

true

Exposure time: 15s

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-TNF alpha antibody [EPR20972] (AB215188)

Immunohistochemical analysis of formalin fixed paraffin embedded rat brain labelling TNF alpha with ab215188 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 [EPR20972] was incubated for 30 mins at room temperature. Sections were counterstained with Hematoxylin. Negative control : no staining on rat brain.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-TNF alpha antibody [EPR20972] (AB215188)

Immunohistochemical analysis of formalin fixed paraffin embedded mouse brain labelling TNF alpha with ab215188 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 [EPR20972] was incubated for 30 mins at room temperature. Sections were counterstained with Hematoxylin. Negative control : no staining on mouse brain.