Overview

  • Product name
    Anti-BDNF antibody [35928.11]
    See all BDNF primary antibodies
  • Description
    Mouse monoclonal [35928.11] to BDNF
  • Host species
    Mouse
  • Tested applications
    Suitable for: ELISA, WB, IHC-Pmore details
  • Species reactivity
    Reacts with: Human
  • Immunogen

    Recombinant human BDNF (rhBDNF) expressed in Sf 21 insect cells.

  • Positive control
    • Brain tissue lysate
  • General notes
    Endotoxin: <10 ng/mg by LAL method.

    For BDNF, multiple WB bands are possible and expected. The human protein has 5 isoforms (precursors: 28 – 37 kDa) and can be glycosylated (Uniprot: http://www.uniprot.org/uniprot/P23560). The mature form is expected at ~14 kDa (monomer) and the dimer at ~28 kDa.

Properties

Applications

Our Abpromise guarantee covers the use of ab10505 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
ELISA Use a concentration of 0.5 - 1 µg/ml.
WB Use a concentration of 1 - 2 µg/ml. Predicted molecular weight: 27.8 kDa.
IHC-P Use at an assay dependent concentration. Perform heat mediated antigen retrieval via the microwave method before commencing with IHC staining protocol.

Target

  • Function
    During development, promotes the survival and differentiation of selected neuronal populations of the peripheral and central nervous systems. Participates in axonal growth, pathfinding and in the modulation of dendritic growth and morphology. Major regulator of synaptic transmission and plasticity at adult synapses in many regions of the CNS. The versatility of BDNF is emphasized by its contribution to a range of adaptive neuronal responses including long-term potentiation (LTP), long-term depression (LTD), certain forms of short-term synaptic plasticity, as well as homeostatic regulation of intrinsic neuronal excitability.
  • Tissue specificity
    Brain. Highly expressed in hippocampus, amygdala, cerebral cortex and cerebellum. Also expressed in heart, lung, skeletal muscle, testis, prostate and placenta.
  • Involvement in disease
    Bulimia nervosa 2
    Congenital central hypoventilation syndrome
  • Sequence similarities
    Belongs to the NGF-beta family.
  • Post-translational
    modifications
    The propeptide is N-glycosylated and glycosulfated.
    Converted into mature BDNF by plasmin (PLG).
  • Cellular localization
    Secreted.
  • Information by UniProt
  • Database links
  • Alternative names
    • Abrineurin antibody
    • ANON2 antibody
    • BDNF antibody
    • BDNF_HUMAN antibody
    • Brain Derived Neurotrophic Factor antibody
    • Brain-derived neurotrophic factor antibody
    • BULN2 antibody
    • MGC34632 antibody
    • Neurotrophin antibody
    see all

References

This product has been referenced in:
  • Jasim H  et al. Saliva as a medium to detect and measure biomarkers related to pain. Sci Rep 8:3220 (2018). Read more (PubMed: 29459715) »
  • Yu J  et al. IL-1ß Stimulates Brain-Derived Neurotrophic Factor Production in Eutopic Endometriosis Stromal Cell Cultures: A Model for Cytokine Regulation of Neuroangiogenesis. Am J Pathol 188:2281-2292 (2018). Read more (PubMed: 30031725) »
See all 6 Publications for this product

Customer reviews and Q&As

1-4 of 4 Abreviews or Q&A

Application
Western blot
Sample
Human Tissue lysate - other (Skeletal muscle)
Gel Running Conditions
Reduced Denaturing (4-15% stain free gel)
Loading amount
20 µg
Specification
Skeletal muscle
Blocking step
Milk as blocking agent for 12 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 6°C

Abcam user community

Verified customer

Submitted Feb 15 2016

Answer

Gracias por contactarnos de nuevo.

¿Podrías por favor indicarme el número de pedido en el que se enviaron los anticuerpos? ¿En qué estado se recibieron? ¿Nada más recibirlos se alicuotaron y guardaron a -20C?

¿En qué aplicación y especies se están usando?

Te agradezco que nos facilites estos detalles para poder entender mejor qué ha podido ocurrir para que ninguno de los tres anticuerpos funcione.

Gracias por la colaboración. Quedo a la espera de tu respuesta.

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Answer

Thank you for contacting us.

I am sorry to hear you have been experiencing problems with Anti-BDNF antibody ab10505 in rat samples.

Unfortunately, this antibody has not been tested with rat samples and therefore we cannot guarantee that it works. Having said this, the sequence similarity of the human BDNF (immunogen) compared to the rat orthologue is 96% and therefore there is a possibility that this antibody could detect rat BDNF.

If you wish, we could have a look at your protocol to work out if there is anything that can be done to improve the results. Therefore, I have attached our questionnaire which will help you put the information we require together very easily.

To my knowledge, it would depend on the tissue type and any kind of treatment whether to expect the mature, plasmin-converted 14.3 kDa form or the 32 kDa pro-BDNF.

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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Question

here the response on your questionnaire. In the second testing, instead of one band at 60 kDa, a lot of bands showed up. Maybe you can identify the problem by looking on our protocol, otherwise I'm open for any suggestions. Thanks in advance, 1. Order details: • Batch number: 174226 • Abcam order or Purchase order number: do not know, order was placed at January, the 18th and antibody arrived at January, the 31th • Antibody storage conditions (temperature/reconstitution etc) Reconstitution: PBS; Storage at -20°C in aliquots 2. Please describe the problem (high background, wrong band size, more bands, no band etc). high background in two of three tests, wrong band size (60 kDa instead of 27,8 kDa) in the other (pictures in the attachment) 3. On what material are you testing the antibody in WB? • Species: human • Cell extract or Nuclear extract: cell extract • Purified protein or Recombinant protein: - 3. The lysate • How much protein was loaded: 50 µg • What lysis buffer was used: RIPA buffer • What protease inhibitors were used: Protease Inhibitor Cocktail (Sigma) • What loading buffer was used: NUPAGE LDS Sample Buffer (Invitrogen) • Did you heat the samples: temperature and time: 95°C for 5 minutes 4. Electrophoresis/Gel conditions/ Transfer conditions • Reducing or non reducing gel: Reducing gel • Gel percentage : 4%-12% • Transfer conditions: Tank Blotting on PVDF membrane 5. Blocking conditions • Buffer: TBS-Tween • Blocking agent: milk, BSA, serum, what percentage: milk, 10% • Incubation time: 1 h • Incubation temperature: room temperature 6. Primary Antibody • Specification (in which species was it raised against): mouse • At what dilution(s) have you tested this antibody: 1:300 • What dilution buffer was used: milk • Incubation time: over night (~18 h) • Incubation temperature: 4°C • What washing steps were done: 3x5’ with TBS-T, 2x5’ with TBS 7. Secondary Antibody • Specification (in which species was it raised against)? rabbit • At what dilution(s) have you tested this antibody: 1:10000 • Incubation time 1 h • Wash steps: 3x5’ with TBS-T, 2x5’ with TBS • Do you know whether the problems you are experiencing come from the secondary? I don’t think so. 8. Detection method ECl, ECl+, other detection method: ECL+ 9. Background bands • Have you eliminated the possibility that any background bands could be due to the secondary antibody? (Run a “No primary” control): Yes • Is the blocking step sufficient? Yes • Are your washing steps sufficiently stringent? (Multiple short washes are more effective than fewer longer wash steps) Yes • At what size are the bands migrating? Could they be degradation products of your target? No degradation product, the bands are slower migrating than the specific product • Please provide an image of your blot (as an e-mail attachment, a faxed image is not sufficient) provided as jpeg 10. Did you apply positive and negative controls along with the samples? Please specify. We applied protein from glioblastoma as positive controls 11. Optimization attempts • How many times have you tried the Western? 3 times • Do you obtain the same results every time e.g. are background bands always in the same place? Yes • What steps have you altered? We took other samples and increased the washing time other incubation with the secondary

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Answer

Thank you very much for providing your protocol details. I think the 60kDa band you originally observed is a result of the homodimerization of BDNF in the samples in your first experiment so this is very promising (for example the datasheet of another BDNF antibody shows banding patterns in rat brain, see the datasheet for ab46176) (https://www.abcam.com/index.html?datasheet=46176). I suspect that further denaturing and reducing of the samples will help the separation of the dimers. It would be good to include a positive control such as brain lysate/hippocampus lysate for example, as levels of BDNF may vary a lot in glioblastoma cells. I suspect that the non specific bands you observe in the next experiments are due to the blocking buffer. I would recommend to incubate the membrane in 5% milk (and to try 5% BSA which in our hands works better) for 1 hour only, and to incubate the antibody overnight at 4C in TBST (Tween 0.1%). If you still experience problems please do not hesitate to contact me,

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