Overview

  • Product nameAnti-TNF alpha antibody [2C8]
    See all TNF alpha primary antibodies
  • Description
    Mouse monoclonal [2C8] to TNF alpha
  • SpecificityThis antibody recognises human TNF-alpha
  • Tested applicationsFlow Cyt, Neutralising, ELISA, IHC-P, IHC-Fr, ICC/IF, WB, Sandwich ELISAmore details
  • Species reactivity
    Reacts with: Mouse, Human
  • Immunogen

    Human recombinant tumor necrosis factor of alpha type

  • Positive control
    • This antibody gave a positive result when used in the following formaldehyde fixed cell lines: A431

Properties

Applications

Our Abpromise guarantee covers the use of ab8348 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Abreviews Notes
Flow Cyt Use 1µg for 106 cells.
Neutralising Use a concentration of 5 µg/ml.
ELISA Use at an assay dependent concentration.
IHC-P Use at an assay dependent concentration.
IHC-Fr Use a concentration of 10 - 20 µg/ml.
ICC/IF Use a concentration of 10 µg/ml.
WB Use at an assay dependent concentration. Predicted molecular weight: 25 kDa.
Sandwich ELISA Use a concentration of 5 µg/ml. Can be paired for Sandwich ELISA with Rabbit polyclonal to TNF alpha (ab9635). Use this antibody as Capture at 5µg/ml with ab9635 as Detection.

Target

  • FunctionCytokine that binds to TNFRSF1A/TNFR1 and TNFRSF1B/TNFBR. It is mainly secreted by macrophages and can induce cell death of certain tumor cell lines. It is potent pyrogen causing fever by direct action or by stimulation of interleukin-1 secretion and is implicated in the induction of cachexia, Under certain conditions it can stimulate cell proliferation and induce cell differentiation.
  • Involvement in diseaseGenetic variations in TNF are a cause of susceptibility psoriatic arthritis (PSORAS) [MIM:607507]. PSORAS is an inflammatory, seronegative arthritis associated with psoriasis. It is a heterogeneous disorder ranging from a mild, non-destructive disease to a severe, progressive, erosive arthropathy. Five types of psoriatic arthritis have been defined: asymmetrical oligoarthritis characterized by primary involvement of the small joints of the fingers or toes; asymmetrical arthritis which involves the joints of the extremities; symmetrical polyarthritis characterized by a rheumatoidlike pattern that can involve hands, wrists, ankles, and feet; arthritis mutilans, which is a rare but deforming and destructive condition; arthritis of the sacroiliac joints and spine (psoriatic spondylitis).
  • Sequence similaritiesBelongs to the tumor necrosis factor family.
  • Post-translational
    modifications
    The soluble form derives from the membrane form by proteolytic processing.
    The membrane form, but not the soluble form, is phosphorylated on serine residues. Dephosphorylation of the membrane form occurs by binding to soluble TNFRSF1A/TNFR1.
    O-glycosylated; glycans contain galactose, N-acetylgalactosamine and N-acetylneuraminic acid.
  • Cellular localizationSecreted and Cell membrane.
  • Information by UniProt
  • Database links
  • Alternative names
    • APC1 antibody
    • APC1 protein antibody
    • Cachectin antibody
    • DIF antibody
    • Differentiation inducing factor antibody
    • Macrophage cytotoxic factor antibody
    • MCF antibody
    • Necrosin antibody
    • TNF a antibody
    • TNF alpha antibody
    • TNF antibody
    • TNF Macrophage Derived antibody
    • TNF Monocyte Derived antibody
    • TNF Superfamily Member 2 antibody
    • TNF superfamily, member 2 antibody
    • TNF, macrophage derived antibody
    • TNF, monocyte derived antibody
    • TNF-a antibody
    • TNF-alpha antibody
    • TNFA antibody
    • TNFA_HUMAN antibody
    • TNFSF2 antibody
    • Tumor necrosis factor alpha antibody
    • Tumor necrosis factor antibody
    • Tumor necrosis factor ligand superfamily member 2 antibody
    • Tumor Necrosis Factor Precursor antibody
    • Tumor Necrosis Factor, Membrane Form antibody
    • Tumor necrosis factor, soluble form antibody
    • Tumour Necrosis Factor Alpha antibody
    see all

Anti-TNF alpha antibody [2C8] images

  • All lanes : Anti-TNF alpha antibody [2C8] (ab8348) (Undiluted)

    Lane 1 : Human skeletal muscle (gastrocnemius) tissue lysate
    Lane 2 : Human skeletal muscle (gastrocnemius) tissue lysate

    Lysates/proteins at 20 µg per lane.

    Secondary
    Alexa Fluor® 555-conjugated Goat anti-mouse IgG polyclonal at 1/5000 dilution

    Performed under reducing conditions.

    Predicted band size : 25 kDa
    Observed band size : 28 kDa (why is the actual band size different from the predicted?)
    Additional bands at : 38 kDa (possible non-specific binding),41 kDa (possible non-specific binding),65 kDa (possible non-specific binding).

    Exposure time : 2 minutes

    This image is courtesy of an anonymous Abreview

    CON+ = Patient with increased TNF alpha levels on ELISA of muscle homogenate.

    POS = Diseased patient with suspected increased TNF alpha levels in muscle.

    See Abreview

  • ab8348 staining TNF alpha in Human skeletal muscle (gastrocnemius) tissue sections by Immunohistochemistry (Formalin/PFA-fixed, paraffin-embedded sections). Tissue was fixed with formaldehyde and blocked with 10% serum for 10 minutes at 25°C; antigen retrieval was by heat mediation in a Tris buffer (pH 8). Samples were incubated with primary antibody (1/200) for 16 hours at 4°C. An undiluted HRP-conjugated Rabbit anti-mouse IgG polyclonal was used as the secondary antibody.

    See Abreview

  • ICC/IF image of ab8348 stained A431 cells. The cells were 4% formaldehyde fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody ab8348 at 10µg/ml overnight at +4°C. The secondary antibody (green) was DyLight® 488 goat anti- mouse (ab96879) IgG (H+L) used at a 1/250 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.

  • Standard curve for TNF alpha (Analyte: ab9642); dilution range 1pg/ml to 1µg/ml using Capture Antibody Mouse monoclonal [2C8] to TNF alpha (ab8348) at 5µg/ml and Detector Antibody Rabbit polyclonal to TNF alpha (ab9635) at 0.5µg/ml.
  • Ab8348 staining human tonsil. Staining is localized to the plasma membrane and cytoplasm.
    Left panel: with primary antibody at 2 ug/ml. Right panel: isotype control.
    Sections were stained using an automated system (Dako PT Link), at room temperature. Sections were rehydrated and antigen retrieved with the Dako 3-in-1 antigen retrieval buffer, EDTA pH 9.0. Slides were peroxidase blocked in 3% H2O2 in methanol for 10 minutes. They were then blocked with Dako Protein block for 10 minutes (containing casein 0.25% in PBS) then incubated with primary antibody for 20 minutes and detected with Dako Envision Flex amplification kit for 30 minutes. Colorimetric detection was completed with diaminobenzidine for 5 minutes. Slides were counterstained with Haematoxylin and coverslipped under DePeX. Please note that for manual staining we recommend to optimize the primary antibody concentration and incubation time (overnight incubation), and amplification may be required.
  • Overlay histogram showing THP1 cells stained with ab8348 (red line). The cells were fixed with 4% paraformaldehyde (10 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab8348, 1µg/1x106 cells) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (ab91353, 2µg/1x106 cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive signal in THP1 cells fixed with 80% methanol (5 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.

  • All lanes : Anti-TNF alpha antibody [2C8] (ab8348) at 1/500 dilution

    Lane 1 : Mouse liver whole tissue lysate
    Lane 2 : Mouse liver whole tissue lysate

    Lysates/proteins at 75 µg per lane.

    developed using the ECL technique

    Performed under reducing conditions.

    Predicted band size : 25 kDa
    Observed band size : 28 kDa (why is the actual band size different from the predicted?)
    Additional bands at : 30 kDa (possible non-specific binding),50 kDa (possible non-specific binding),65 kDa (possible non-specific binding).

    Exposure time : 15 minutes

    This image is courtesy of an anonymous Abreview

    See Abreview

Protocols

References for Anti-TNF alpha antibody [2C8] (ab8348)

This product has been referenced in:
  • af Klint E  et al. Intraarticular glucocorticoid treatment reduces inflammation in synovial cell infiltrations more efficiently than in synovial blood vessels. Arthritis Rheum 52:3880-9 (2005). Read more (PubMed: 16320336) »
  • Benyo DF  et al. Hypoxia stimulates cytokine production by villous explants from the human placenta. J Clin Endocrinol Metab 82:1582-8 (1997). Read more (PubMed: 9141553) »

See all 2 Publications for this product

Product Wall

Abcam guarantees this product to work in the species/application used in this Abreview.
Application Immunocytochemistry/ Immunofluorescence
Blocking step Serum as blocking agent for 20 minute(s) · Concentration: 10% · Temperature: 25°C
Sample Human Tissue (Skeletal Muscle (Gastrocnemius))
Specification Skeletal Muscle (Gastrocnemius)
Permeabilization No
Fixative Methacarn
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Submitted Aug 02 2013

Application Western blot
Loading amount 75 µg
Gel Running Conditions Reduced Denaturing (Gradient Gel)
Sample Mouse Tissue lysate - whole (Liver)
Specification Liver
Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 3% · Temperature: RT°C
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Submitted Mar 20 2014

Application Immunoprecipitation
Immuno-precipitation step Protein A/G
Sample Mouse Cell lysate - nuclear (mouse ES cells)
Specification mouse ES cells
Total protein in input 50 µg
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Submitted Feb 18 2014

Application Immunoprecipitation
Immuno-precipitation step Protein A/G
Sample Human Cell lysate - nuclear (HCT116)
Specification HCT116
Total protein in input 100 µg
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Submitted Feb 18 2014

Application Western blot
Loading amount 50 µg
Gel Running Conditions Reduced Denaturing (10%)
Sample Human Cell lysate - whole cell (Breast cancer line - MDAMB231)
Specification Breast cancer line - MDAMB231
Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 3% · Temperature: rT°C
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Submitted Dec 30 2013

Application Immunoprecipitation
Immuno-precipitation step Other - Protein G Dynabeads
Sample Human Cell lysate - whole cell (Breast cancer lines - MDAMB231, and few others (re)
Specification Breast cancer lines - MDAMB231, and few others (re
Total protein in input 75 µg
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Submitted Dec 27 2013

Abcam guarantees this product to work in the species/application used in this Abreview.
Application Western blot
Loading amount 20 µg
Gel Running Conditions Reduced Denaturing (12% Bis-Tris)
Sample Human Tissue lysate - whole (Skeletal Muscle (Gastrocnemius))
Specification Skeletal Muscle (Gastrocnemius)
Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C
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Submitted Aug 02 2013

Abcam guarantees this product to work in the species/application used in this Abreview.
Application Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Blocking step Serum as blocking agent for 10 minute(s) · Concentration: 10% · Temperature: 25°C
Antigen retrieval step Heat mediated - Buffer/Enzyme Used: Tris (pH8) better than Citrate (pH6)
Sample Human Tissue sections (Skeletal Muscle (Gastrocnemius))
Specification Skeletal Muscle (Gastrocnemius)
Permeabilization No
Fixative Formaldehyde
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Submitted Aug 01 2013

Thank you for contacting us.

Here is the information about our Abtrial program.

For UNTESTED species and/or applications, we have established a testing discount program. Here is a brief description of how it works:

The testin...

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Thank you for contacting us.

We have in our catalogue the following pairs of antibodies that have been tested for the detection of Human TNF-alpha in sELISA :

Anti-TNF alpha antibody [2C8] (http://www.abcam.com/ab8348) as capture + Ra...

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Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"