Overview

  • Product name
  • Description
    Rabbit polyclonal to TNF alpha
  • Host species
    Rabbit
  • Tested applications
    Suitable for: IHC-Fr, IHC-P, WB, Neutralising, Sandwich ELISA, ICC/IFmore details
  • Species reactivity
    Reacts with: Mouse, Human
  • Immunogen

    Recombinant fragment corresponding to Mouse TNF alpha aa 80-235. E.coli-derived recombinant MurineTNF-alpha
    Database link: P06804

  • Positive control
    • Recombinant mouse TNF alpha protein (ab9740) can be used as a positive control in WB. This antibody gave a positive result in IF in the following Formaldehyde fixed cell line: A431

Properties

Applications

Our Abpromise guarantee covers the use of ab9739 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
IHC-Fr Use at an assay dependent concentration. PubMed: 18458097
IHC-P Use at an assay dependent concentration.
WB Use at an assay dependent concentration.

To detect TNF alpha by Western Blot analysis this antibody can be used at a concentration of 0.1 - 0.2 µg/ml. Used in conjunction with compatible secondary reagents the detection limit for recombinant TNF alpha is 1.5 - 3.0 ng/lane, under either reducing or non-reducing conditions. The presursor is ~26 kDa and the secreted form is ~17 kDa.

Neutralising Use at an assay dependent concentration. To yield one-half maximal inhibition [ND50] of the biological activity of mTNF-alpha (0.25 ng/ml), a concentration of 0.08 – 0.10 µg/ml of this antibody is required.
Sandwich ELISA Use at an assay dependent concentration.

To detect Murine TNF alpha by sandwich ELISA (using 100 μl/well antibody solution) a concentration of 0.5 - 2.0 μg/ml of ab9739 is required. This antigen affinity purified antibody, in conjunction with a suitable detection antibody, allows the detection of at least 0.2 - 0.4 ng/well of recombinant Murine TNF alpha.  

ICC/IF Use a concentration of 5 µg/ml.

Target

  • Function
    Cytokine 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 disease
    Genetic 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 similarities
    Belongs 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 localization
    Secreted 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
    • Tnf 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, Membrane Form antibody
    • Tumor necrosis factor, soluble form antibody
    see all

Images

  • All lanes : Anti-TNF alpha antibody (ab9739) at 1/3500 dilution

    Lane 1 : 100ug LPS stimulated J774.A1 mice macrophages
    Lane 2 : 50ug LPS stimulated J774.A1 mice macrophages
    Lane 3 : 25ug LPS stimulated J774.A1 mice macrophages

    Secondary
    All lanes : HRP conjugated goat anti-rabbit antibody

    Performed under reducing conditions.

    Observed band size: 17 kDa
    why is the actual band size different from the predicted?

    See Abreview

  • ab9739 (2µg/ml) staining TNF alpha in human tonsil using an automated system (DAKO Autostainer Plus). Using this protocol there is strong cytoplasmic staining within the germinal follicle.
    Sections were rehydrated and antigen retrieved with the Dako 3 in 1 AR buffer EDTA pH 9.0 in a DAKO PT Link. Slides were peroxidase blocked in 3% H2O2 in methanol for 10 mins. They were then blocked with Dako Protein block for 10 minutes (containing casein 0.25% in PBS) then incubated with primary antibody for 20 min 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, optimization of primary antibody concentration and incubation time is recommended. Signal amplification may be required.
  • ICC/IF image of ab9739 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 ab9739 at 5µg/ml overnight at +4°C. The secondary antibody (green) was DyLight® 488 goat anti- rabbit (ab96899) IgG (H+L) used at a 1/1000 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.

  • Immunohistochemical analysis of colchicine injected mouse brain (hippocampus CA1 region) tissue using ab9739 at 1.0 μg/ml overnight at 4˚C. This was followed by a peroxidase conjugated secondary antibody and then a fluorescein Tyramide Signal Amplification (TSA™) reagent.  Optimal concentrations and conditions may vary.

References

This product has been referenced in:
  • Alhasson F  et al. High circulatory leptin mediated NOX-2-peroxynitrite-miR21 axis activate mesangial cells and promotes renal inflammatory pathology in nonalcoholic fatty liver disease. Redox Biol 17:1-15 (2018). IHC-P ; Mouse . Read more (PubMed: 29660503) »
  • Gao JY  et al. Depressive- and anxiety-like phenotypes in young adult APPSwe/PS1dE9 transgenic mice with insensitivity to chronic mild stress. Behav Brain Res 353:114-123 (2018). Read more (PubMed: 30012417) »
See all 48 Publications for this product

Customer reviews and Q&As

1-10 of 23 Abreviews or Q&A

Application
Western blot
Sample
Mouse Tissue lysate - whole (Mouse lung)
Gel Running Conditions
Reduced Denaturing
Loading amount
15 µg
Treatment
Mouse lung after 24-hour ischemia
Specification
Mouse lung
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 23°C

Abcam user community

Verified customer

Submitted Mar 03 2017

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunohistochemistry (Frozen sections)
Sample
Mouse Tissue sections (breast tumor)
Permeabilization
No
Specification
breast tumor
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 20°C
Fixative
Formaldehyde

Abcam user community

Verified customer

Submitted Feb 15 2017

Application
Western blot
Sample
Mouse Serum (Mouse Serum)
Gel Running Conditions
Reduced Non-Denaturing (Native)
Loading amount
20 µg
Specification
Mouse Serum
Blocking step
Odyssey Blocking Buffer as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 100% · Temperature: 26°C

Abcam user community

Verified customer

Submitted Jul 30 2014

Application
Western blot
Loading amount
20 µg
Gel Running Conditions
Reduced Denaturing
Sample
Mouse Cell lysate - whole cell (hepatocytes)
Specification
hepatocytes
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 23°C

Abcam user community

Verified customer

Submitted Jul 08 2014

Answer

Thank you for contacting us.

Unfortunately, we cannot test every organ or tissue when we establish a product.However, if the knee joints were decalcified properly and the species is tested, I don't see any problems why these antibodies shouldn't work in these sections.

Our policy is that we are happy to offer a refund, credit note or free of charge replacement when a product is not working in a successfully tested applications or species (and the product has been purchased in the last 180 days).

I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

Read More

Answer

All of these antibodies recognise cytokines which are present in inflammed tissue therefore any inflammed tissue would be a good positive control.

In our catalogue, we have several cancer slides which would be appropriate:
tonsil: (ab5146)
breast (ab4701)
pancreatic (ab5036)
stomach (ab5115)
colon (ab4702)

Alternatively, spleen tissues seem to be a very good positive control for these cytokines and are used on several of our other datasheets.

I would suggest using ab4372 if you are working on human samples, ab4613 if you are working on mouse samples or ab4624 if you are working on rat samples.

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Answer

Vielen Dank für Ihre Anfrage.

Ich habe vier primäre AK für Sie herausgesucht, die alle in einer relativ hohen Verdünnung eingesetzt werden können, so dass sie für eine Weile reichen sollten. Das Sie alle Proben nun damit verarbeitet bekommen kann ich leider nicht versprechen, da die Antikörper ja auch in den meisten Fällen ein paar Optimierungsexperimente benötigen.

https://www.abcam.com/index.html?datasheet=9739 (or use the following: https://www.abcam.com/index.html?datasheet=9739).

https://www.abcam.com/index.html?datasheet=9969 (or use the following: https://www.abcam.com/index.html?datasheet=9969).

https://www.abcam.com/index.html?datasheet=13243 (or use the following: https://www.abcam.com/index.html?datasheet=13243).

https://www.abcam.com/index.html?datasheet=83339 (or use the following: https://www.abcam.com/index.html?datasheet=83339).


Als Sekundär-AK würde ich Ihnen den ab97069 Goat anti-Rabbit IgG H&L (HRP) secondary antibody empfehlen, da dieser bis zu einer Verdünnung von 1/50.000 eingestzt werden kann.



https://www.abcam.com/index.html?datasheet=97069 (or use the following: https://www.abcam.com/index.html?datasheet=97069).


Ich hoffe, dies hilft Ihnen weiter. Bitte zögern Sie nicht, sich wieder bei uns zu melden, falls Sie weitere technische Fragen haben. Falls Sie die Produkte Bestellen möchten oder mehr Informationen zB bezüglich des Preises wünschen, wenden Sie sich einfach an unseren Kundendienst.

Benutzen Sie unsere Produkte? Schicken Sie uns einen Abreview. Verdienen Sie sich eine Belohnung!
https://www.abcam.com/abreviews

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Answer

Vielen Dank für Ihren Anruf und für Ihr Interesse an unseren Produkten.

Unserem Wissen nach wurde ab17442 bisher noch nicht in sandwichELISA getestet. Falls Sie dies selbst testen möchten, kann ich Ihnen zurzeit ein spezielles Angebot über einen 100%igen Abreview-Rabatt anbieten. Bei diesem Angebot bekommen Sie einen Rabatt für eine zukünftige Bestellung, wenn Sie uns ein Abreview mit dem Testresultat zusenden. Der Rabatt würde den ganzen Wert von ab17442 abdecken, und gegen eine erneute Bestellung eines weiteren Produkts von uns verrechnet werden.

Um von diesem Angebot profitieren zu können, folgen Sie bitte diesen Schritten:

1.) Bestätigen Sie mir bitte, dass Sie ab17442 in sandwichELISA testen möchten.

2.) Bitte bestellen Sie erst nach Erhalt des Rabattcodes.

3.) Bestellen Sie den Antikörper wie üblich per Telefon, Email oder Fax

4.) Testen Sie den Antikörper in sandwichELISA.

5.) Teilen Sie uns das Ergebnis Ihres Tests durch ein Abreview mit und notieren Sie den Discount Code in dem Feld "additional notes"
Unter der folgenden URL können Sie mehr über unser Abreview System erfahren:
https://www.abcam.com/abreviews

6.) Der Rabattcode ist nach dem Abschicken des Abreviews aktiv, und Sie können einen anderen Produkt zu dem gleichen Preis wie ab17442 bei uns bestellen (halten Sie bei der Bestellung bitte den Rabattcode und die Bestellnummer von ab17442 bereit). Bitte beachten Sie, dass der Rabattcode innerhalb von 4 Monaten nach Ausstellung eingelöst werden muss.

Der Rabattcode wird gültig unabhängig davon, ob Ihr Ergebnis positiv oder negativ ist.

Die Bedingungen zu unserem 100% Abreview Rabatt können Sie unter dem folgenden Link nachlesen:
https://www.abcam.com/collaborationdiscount

Dieses Angebot kann ich Ihnen auch für ab16166 machen, falls Sie einen monoklonalen Detektionsantikörper bevorzugen.

https://www.abcam.com/index.html?datasheet=16166 (or use the following: https://www.abcam.com/index.html?datasheet=16166).


Ich hoffe, diese Informationen helfen Ihnen weiter. Falls Sie einen Rabattcode erhalten möchten oder weitere Fragen haben, zögern Sie bitte nicht, sich wieder an mich zu wenden.

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Answer

Thank you for contacting us and for your interest in our products.

As far as we are aware, ab9739 has never been tested in IP. All tested applications covered by the 6 month guarantee are specified on our datasheets, and these are updated as soon as any new information is brought to our attention.

If you are running samples in WB that having been purified using IP, I can suggest that the band you are seeing at 55 kDa is probably the Ig heavy chain which has eluted from the beads with the protein during purification. This is quite common. To enable elution of protein with little antibody contamination (for cleaner protein preparation and cleaner western blots), it is recommended to cross link the antibody to the beads. An example procedure for this is provided on the follow page from our protocols section on the website. The target protein should then be eluted with a mild eluent, such as glycine buffer. I hope this will be helpful to you.

https://www.abcam.com/index.html?pageconfig=resource&rid=11415

If you are obtaining a band at 55 kDa in WB of normal cell lysate samples, please do not hesitate to contact me with further information in the questionaire below and I will be pleased to investigate this further for you. The antibody is covered by the 6 month guarantee in WB.

As you are using IP, we like to encourage all of our customers to submit an Abreview via the online product datasheet. We always appreciate customer feedback, whether positive or negative, and we make all product information available to researchers. Plus, each Abreview earns Abpoints that can be used for discounts on future purchases or rewards such as Amazon.com gift certificates.

To find out more about our Abreview system, please see the following link:

https://www.abcam.com/abreviews

I hope this information is helpful. Please do not hesitate to contact me for any further advice or information.

Order Details
Antibody code:

Problem
Choose: Non-specific band Multiple bands No signal or weak signal High background

Lot number

Purchase order number
or preferably Abcam order number:



General Information
Antibody storage conditions (temperature/reconstitution etc)


Description of the problem (high background, wrong band size, more bands, no band etc.)


Sample (Species/Cell extract/Nuclear extract/Purified protein/Recombinant protein etc.)


Sample preparation (Buffer/Protease inhibitors/Heating sample etc.)


Amount of protein loaded


Electrophoresis/Gel conditions (Reducing or Non-reducing gel, % of the gel etc.)


Transfer and blocking conditions (Buffer/time period, Blocking agent etc.)


Primary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, Wash step)


Secondary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, Wash step)


Detection method (ECL, ECLPlus etc.)


Positive and negative controls used (please specify)



Optimization attempts (problem solving)
How many times have you tried the Western?



Have you run a "No Primary" control?
Yes No

Do you obtain the same results every time?
Yes No
e.g. are the background bands always in the same place?


What steps have you altered?


Additional Notes:


Image:
We would appreciate if you are able to provide an image (including molecular weight markers) which would help us to asess the results.

Read More

Answer

Thank you very much for your reply.

You are correct, and I apologize for this mistake. I converted the 100 micrograms into milligrams, not grams as it should have been for the calculation. There should be 4.01e14 molecules per vial. Thank you for spotting that error and pointing it out.

Please let me know if you have any questions or need anything further. Have a nice day!

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1-10 of 23 Abreviews or Q&A

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