Anti-TNF alpha antibody [RM1005]

10 product images

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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 1?g/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 (Rabbit IgG, monoclonal [EPR25A] - Isotype Control 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, Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed ab150081) at 1/2000 dilution was used as the secondary antibody.

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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, Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed 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.

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  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) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/100000 dilution

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

  Exposure time: 180s

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  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)(VeriBlot for IP Detection Reagent (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: abAB307164 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 (Rabbit IgG, monoclonal [EPR25A] - Isotype Control 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

  Lane 3: Immunoprecipitation - Rabbit IgG, monoclonal [EPR25A] - Isotype Control (Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730)

  Secondary

  All lanes: Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (VeriBlot for IP Detection Reagent (HRP) ab131366) at 1/5000 dilution

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

  Exposure time: 6s

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  Immunohistochemistry - 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

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  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)(VeriBlot for IP Detection Reagent (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: abAB307164 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 (Rabbit IgG, monoclonal [EPR25A] - Isotype Control 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

  Lane 3: Immunoprecipitation - Rabbit IgG, monoclonal [EPR25A] - Isotype Control (Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730)

  Secondary

  All lanes: Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (VeriBlot for IP Detection Reagent (HRP) ab131366) at 1/5000 dilution

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

  Exposure time: 6s

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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 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed 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 ?g/ml) for 3 hours.Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8). Alexa Fluor® 594 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker 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 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor? 488) preadsorbed at 1/1000 (2 ug/ml) dilution.

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  Immunohistochemistry - 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

• 

  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 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed 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). Alexa Fluor® 594 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker 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 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor? 488) preadsorbed at 1/1000 (2 ug/ml) dilution.

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  Indirect ELISA - Anti-TNF alpha antibody [RM1005] (ab307164)

  Indirect ELISA analysis of Anti-FGFR1 (phospho Y653) antibody [EPR843(N)] 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).