Overview

Properties

Applications

Our Abpromise guarantee covers the use of ab9579 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 1-2µg for 106 cells.

ab170190 - Mouse monoclonal IgG1, is suitable for use as an isotype control with this antibody.

 

WB Use at an assay dependent concentration.
IHC-P 1/100. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
IHC-Fr Use at an assay dependent concentration.
ICC/IF Use at an assay dependent concentration.

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

  • The antibody ab9579 labels the cytoplasm of TNF-a secreting cells including histiocytes/macrophages.
  • Overlay histogram showing THP1 cells stained with ab9579 (red line). The cells were fixed with 80% methanol (5 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 (ab9579, 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 4% paraformaldehyde (10 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.

  • Immunohistochemisty of Human adenocarcinoma uterum (with positive reactive macrophages).
  • Immunochistochemistry of Human mandibular cyst (granulomatous inflammation with positive macrophages).

References

This product has been referenced in:
  • Deleage C  et al. Impact of early cART in the gut during acute HIV infection. JCI Insight 1:N/A (2016). IHC-P ; Human . Read more (PubMed: 27446990) »
  • Thu VT  et al. NecroX-5 exerts anti-inflammatory and anti-fibrotic effects via modulation of the TNFa/Dcn/TGFß1/Smad2 pathway in hypoxia/reoxygenation-treated rat hearts. Korean J Physiol Pharmacol 20:305-14 (2016). Read more (PubMed: 27162485) »
See all 15 Publications for this product

Customer reviews and Q&As

1-10 of 10 Abreviews or Q&A

Answer

Thank you for your enquiry. I have attached a copy of the IHC image that is on the datasheet for the antibody, a granulomatous inflammatory reaction, probably in a chronic cholecystitis. The species is human. Please let me know if you have any questions.

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Question


Customer purchased the same antibodies (ab49679, ab66184, ab74525)in two different moments from different batches and she is complaining that they are showing too many differences between batches in terms of optimal working dilutions. All antibodies acquired in the fisrt purchase showed good staining in higher dilutions than the antibodies acquired in the second purchase. Customer is unsatisfied with these batch variations.


Regarding the antibodies ab9579 and ab9542 customer is complaining that they showed good staining in the first usages, but after four months they are not reacting anymore.

She said her protocol is always the same, find it described below. And please, find attached a file containing imageswith better description of the problem.


Abcam IHC Questionnaire

1) Abcam product code: ab49679, ab66184, ab74525, ab9579, ab9542

2) Lot number: customer does not have the lot numbers because she aliquoted the antibodies.

3) Antibody storage conditions (temperature/reconstitution etc): ab49679, ab66184, ab74525, and ab9542 were aliquoted and stored at -20°C. Ab9579 is stored at 4°C.

4) Description of the problem:

ab49679, ab66184 and ab74525 were purchased in two different moments and customer is complaining they are showing big differences between batches in terms of optimal working dilutions. All antibodies acquired in the first purchase showed good staining in higher dilutions than the antibodies acquired in the second purchase. The antibodies acquired in the second purchase are showing very weak staining in very low dilutions like 1:10.

ab9579 and ab9542 showed good staining in the first usages, but after four months they are not reacting anymore.

5) Sample preparation
Species: human

Type of sample (Fresh frozen sections, perfusion fixed frozen sections; PFA/formalin fixed paraffin embedded sections, cells in culture, other): formalin fixed paraffin embedded sections

6) Fixation step
Yes/No: Yes

If yes:
Fixation time:
Fixation temperature:

7) Antigen retrieval method: Trilogy solution from Cell Marque in pressure cooker for 15 min. (http://www.cellmarque.com/pdf_files/direct/160.pdf)


8) Permeabilization method
Did you do a permeabilization step (details please) or add permeabilizing agent in any dilution buffers? No
Permeabilizing agent and concentration:


9) Blocking step: Background Block solution from Cell Marque for 10 min (http://www.cellmarque.com/pdf_files/direct/174.pdf)

10) Endogenous peroxidases blocked? Yes, Peroxide Block solution for 10 min at RT.

11) Primary antibody (If more than one was used, describe in “additional notes”):
ab49679 – diluted at 1:100 and 1:50, overnight incubation –> very weak staining, almost negative.
ab66184 – diluted at 2,5 ug/ml, overnight incubation –> very weak staining, almost negative.
ab74525 – diluted at 1:20 and 1:10, overnight incubation –> to have a good staining we used the antibody almost undiluted.
ab9579 – diluted at 1:100, overnight incubation –> no staining
ab9542 – diluted at 1:50, overnight incubation –> no staining

12) Secondary antibody: HiDef Detection™ HRP Polymer System from Cell Marque (http://www.cellmarque.com/pdf_files/direct/2041.pdf)

Incubation time: 20 min at 37°C.

13) Washing after primary and secondary antibodies: Yes
Buffer: PBS
Number of washes: 3

14) Detection method: DAB

15) How many times have you run this staining? 3
Do you obtain the same results every time? Yes
What steps have you altered to try and optimize the use of this antibody? Primary antibody dilutions and incubation time.

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Answer

Thank you for your inquiry.

I am sorry to hear that your customer is having trouble with these antibodies.

It seems to me that there must be an issue with the HRP polymer detection system, DAB,or antigen retrieval buffers/system. The reason is that Iooked up the previous Sellex orders for this product and the customer is comparing the same lots with the old and new batches for two products.

For ab49679, CD105, the customer is having trouble with lot GR41983-2. The previous lot that worked for them on the earlier Sellex order was GR41983-1. These are the same lots, except the -2 was aliquoted after the -1.

For ab66184, VEGFR1, the customer is having trouble with lot GR30735-3. The previous lot that worked for them on the earlier Sellex order was GR30735-2. Again, these are the same lots, except the -3 was aliquoted after the -2.

For ab74525, TGF beta-1, the cutomer was having trouble with lot GR69795-1. This lot is different than the GR14885-2 lot that worked well for them on the earlier Sellex order. So there may be a lot variation in this instance, but I would recommend using all new buffers in the protocol to ensure this.

Both ab9542 and ab9579 were purchased on the same order back in June 2011, which is outside our 6 month Abpromise period.

There must be something going on with the customer's detection system, buffers, or perhaps they are using a frost-free freezer, or thefreezers went down,etc. It wouldn't make sense for all 5 antibodies to show decreasing signal, requiring more antibody. Especially because they are using 2 of the same lots for 2 antibodies, there can't be lot-to-lot variations in these cases.

I hope this information helps. Please contact us with any other questions.

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Question
Answer

Thank you for contacting us. I have received the following information from the lab: The antigen retrieval performed with this antibody in paraffin sections is: 5 minutes heat-induced epitope retrieval (HIER) in boiling citrate buffer, pH 6.0. I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

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Answer

Thank you for your enquiry. From the three antibodies that you list I would like to recommend TNF alpha antibody (ab6671). This antibody has received a favourable Abreview from one of our customers detailing its application following citrate antigen retrieval. I hope this information helps, please do not hesitate to contact us if you need any more advice or information.

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Question
Answer

Thank you for your enquiry. The best positive control for this antibody is human macrophages in a granulomatous inflammatory lesion. I hope this information helps. Please do not hesitate to contact us if you need anything further.

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Answer

Thank you for your enquiry. I am sorry to hear that you have been experiencing problems with ab8999. As discussed on the telephone this afternoon please would you fill in the questionnaire by clicking on the link below. I would also appreciate it if you could send me details of the LOT number and images of the blots for both ab8999 and the anti-Prp (ab3408) that we discussed. This will give me an appreciation of what you have done to optimise this antibody by western blot. I hope that we can resolve this problem rapidly. https://www.abcam.com/index.html?section=western&pageconfig=technical&intAbID=8999&mode=questionaire I look forward to hearing from you.

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Answer

Thank you for your email. Did you order the antibody directly from Abcam or from one a distributor, such as Biozol Diagnostica? If you ordered from a distributor, please indicate which one and also the purchase order number that you used with them. The replacement will then be shipped to you via the distributor. I also received the following information regarding the Western blot protocol that was used when the antibody was tested which I wanted to pass on to you: "Sample preparation 0.5% Triton-X100 containing PBS with protease inhibitors (1mMPMSF, 1ug/ml aprotinin and leupeptin) 45 min on ice. Sample buffer for SDS-ELFO Boiling for 5 min in 0.5M Tris-HCl buffer contain 10% SDS and 1% mercaptoethanol, 25% glycerol ELFO Separation on 10% polyacrylamide gels according to Laemmli (Running with 200V). Immunoblot Blotting to nitrocellulose sheets (Schleicher & Schuell) overnight at 35mA. Blocking with 5% non-fat milk powder for 1 hour then incubation with anti-TNF-? mAbs (1-5 ug/ml) for 1 hr, then HRP conjugated anti-mouse antibodies used as second reagent (Dako) for 1 hr. Development with ECL reagents (Amersham). PBS containing 0.05% Tween-20 for washing among the reaction steps."

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Answer

Thank you for your message. I have checked with the originator of this antibody and there have not been any complaints concerning this lot. I'm still waiting to obtain some more information regarding the use of this antibody in Western blotting, but I can send you a replacement lot to try if you wish. Please let me know and also when you would like it shipped as many places will be closed over the holidays. Thank you, and happy holidays!

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Answer

Thank you for your enquiry and I'm sorry to hear that you are experiencing difficulty with this antibody. I would like to make a few suggestions to try to help you out. To see if you can obtain a signal, try incubating with the primary antibody overnight at 4C. Make sure that the protein transferred properly to the membrane (check this by using Ponceaue S). Also, increase the concentration of the secondary antibody. Is the secondary antibody working with other primaries? You may also want to decrease your number of washes. If you still are not seeing a signal, please do let me know.

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Answer

Yes, ab9570 will work using Envision + mouse and/or Envision mouse-rabbit kit.

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

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