Overview

  • Product name
    Anti-DEFB-4 antibody [L13-10-D1]
    See all DEFB-4 primary antibodies
  • Description
    Mouse monoclonal [L13-10-D1] to DEFB-4
  • Host species
    Mouse
  • Tested applications
    Suitable for: ELISA, WB, RIA, IHC-Pmore details
  • Species reactivity
    Reacts with: Human
  • Immunogen

    Synthetic peptide corresponding to Human DEFB-4 aa 25-61.
    Sequence:

    ELDRICGYGTARCRKKCRSQEYRIGRCPNTYACCLRK)


    Database link: Q8WTQ1
    (Peptide available as ab69499)

  • Positive control
    • IHC-P: Uterus and kidney sections.
  • General notes

    Protein previously labeled as beta 4 Defensin.

Properties

Applications

Our Abpromise guarantee covers the use of ab14419 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 at an assay dependent concentration.
WB Use at an assay dependent concentration. Predicted molecular weight: 8 kDa.Can be blocked with Recombinant human DEFB-4 protein (ab69499).
RIA Use at an assay dependent concentration.
IHC-P Use at an assay dependent concentration.

Target

  • Function
    Has antimicrobial activity. Synergistic effects with lysozyme and DEFB103.
  • Tissue specificity
    High expression in the testis. Gastric antrum exhibited relatively high levels. A lower expression is observed in uterus and neutrophils thyroid gland, lung, and kidney. No detectable expression in other tissues tested.
  • Sequence similarities
    Belongs to the beta-defensin family.
  • Cellular localization
    Secreted.
  • Information by UniProt
  • Database links
  • Alternative names
    • BD 4 antibody
    • BD-4 antibody
    • BD4 antibody
    • beta 104 antibody
    • beta 4 Defensin antibody
    • Beta defensin 4 antibody
    • Beta-defensin 104 antibody
    • Beta-defensin 4 antibody
    • D104A_HUMAN antibody
    • DEFB-4 antibody
    • DEFB104B antibody
    • Defensin antibody
    • Defensin beta 104 antibody
    • Defensin beta 4 precursor antibody
    • hBD 4 antibody
    • hBD-4 antibody
    • hBD4 antibody
    see all

Images

  • ab14419 staining DEFB-4 in human pancreatic tissue by Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections). Tissue was fixed with formaldehyde and a heat mediated antigen retrieval step was performed. Samples were then incubated with the primary antibody at a 1/100 dilution for 1 hour at 25°C. An undiluted HRP-conjugated goat IgG polyclonal was used as the secondary antibody.

    See Abreview

References

This product has been referenced in:
  • Dréno B  et al. Hidradenitis Suppurativa: The Role of Deficient Cutaneous Innate Immunity. Arch Dermatol : (2011). ICC/IF ; Human . Read more (PubMed: 22004878) »
  • Garreis F  et al. Roles of human beta-defensins in innate immune defense at the ocular surface: arming and alarming corneal and conjunctival epithelial cells. Histochem Cell Biol 134:59-73 (2010). IHC-P ; Human, Mouse . Read more (PubMed: 20526610) »
See all 2 Publications for this product

Customer reviews and Q&As

1-4 of 4 Abreviews or Q&A

Question

The numbers are based on the expected concentrations after reconstitution. Customer said she can perform a Bradford assay to confirm, no problem. But she is still worried cause she is having no bands. She would like us to find a reason why there is no bands in WB and there is some spots in dot-blot? She asked if these could be related to the gel condition (which is denaturing)? Also, these antibodies detect the denatured form or native form of proteins/standards?



Regarding ab14425 (beta defensin 1 antibody), I read the following on the support page

Question 6



Thursday 03-November-2005









In response to your enquiry, here is what Autogen Bioclear wrote:

"The beta-defensin genes were directly expressed (no tags or leader sequence). Sequences are available (let me know if you require them). After fermentation, centrifugation, cell breakage; the proteins are folded into its active form during the folding/oxidation stage, this is when the disulfide bridges form. Specific processes are proprietary. The beta-defensins have a characteristic 6 cysteine structural motif containing 3 disulfide-bridges."

I have tested the performance of your antibody against the denatured forms of my standards, HBD-1 (36aa and 47aa), HBD-2 and HBD-3. Unfortunately these results were negative and the only positive results were the native forms of HBD-1 36 and 47 aa, which gave a clear signal on the dot blot.

Best regards







ANSWER:





Thank you for getting back to me with those details, it was very interesting.

Unfortunately given that the source of the antisera have not been able to provide me with further details as to the synthesis of the immunising peptides it is difficult for me to comment on whether they have been raised against a correctly folded immunogen.

If this was indeed the case then one might speculate that these antibodies only recognise the synthetic peptide and not the native form of the protein. However, checking back to past orders and this antibody is relatively popular and we have not received any complaints as such. I can but speculate that the absence of western or dot blot as applications on the datasheets is a reflection that these antibodies do not recognise their epitopes when immobilised on a membrane.










What do you think might be the problem with these beta defensins?



Do you have some more useful information about these specific antibodies/antigens conditions of binding ?



http://www.uniprot.org/uniprot/P60022 (Molecular mass is 3928±0.5 Da from positions 33 - 68.)

http://www.uniprot.org/uniprot/O15263

http://www.uniprot.org/uniprot/P81534 (Molecular mass is 5154.59 Da from positions 23 - 67.)

http://www.uniprot.org/uniprot/Q8WTQ1






I am looking forward to hearing from you. We need to solve this case as soon as possible because time is passing by and customer has a deadline to finish her experiments with beta defensins.

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Answer

Has our customer had a chance to test the protein concentrations via Bradford assay?

I have unfortunately been unable to locate western blot data. Based on the information it may be that a native western blot system is required. It is also possible these antibodies are not working as expected.

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Answer

Thank you for sending along the dot blots and additional information. We really appreciate the extra troubleshootingeffort.

Here are my thoughts:

1. In general it looks like the antibodies are able to detect Defensin in the chorioamniotic membrane samples.

2. It does not appear that the antibodies detected recombinant Defensin protein. You note the mass of recombinant protein spotted on the membranes, from 400ng - 0.78ng. Was the concentration of the Defensin proteinconfirmed by Bradford assay or OD280n measurements following reconstitution or are these number based on the expected concentrations after reconstitution?

If the concentrations are only theoretical, could you please check the concentrations to ensurethey are accurate?

Thanks again for your troubleshooting efforts. I'm sure we'll be able to resolve this situation soon.

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Question

For the technical support,   Please, customer is having problems using human beta defensin antibodies and their respective recombinant proteins in WB applications.   Find bellow the technical questionnaire filled out by the customer.   As the beta defensins are small proteins (8 kDa), it seems this might be the origin of the problem. Maybe, the customer is losing these small bands during the running or even during the transfer. Could you please verify her protocol and give suggestions on how to solve this problem?   Thank you in advance.   Kind Regards, Abcam WB Questionnaire   1) Abcam product code ab14425 + ab50048 ab66072 + ab9872 ab19270 + ab50059 ab14419 + ab69499                            2) Lot number ab14425: GR 17872-2 + ab50048: GR 45073-1 ab66072: GR 40785-1 + ab9872: GR 11654-6 ab19270: GR 730-1 + ab50059: GR 1609-2 ab14419: GR 21054-1 + ab69499: GR 3598-2     3) Antibody storage conditions (temperature/reconstitution etc) Antibodies: stored at -20 ºC. Recombinant proteins: after adding 100 ul PBS + 15% glycerol we aliquoted each one and stored at -80°C.   4) Description of the problem (high background, wrong band size, more bands, no band etc.) No bands using the beta defensin antibodies with their respective recombinant protein and no bands with lysate of chorioamniotic membrane.   5) Sample (Species/Cell extract/Nuclear extract/Purified protein/Recombinant protein etc.) Human recombinant proteins and lysate of chorioamniotic membrane tissue from human.   6) Sample preparation (Buffer/Protease inhibitors/Heating sample etc.) Lysis buffer: - 50mM Tris HCl pH 7,4 - 0,2mM NaCl - 0,1% Triton X-100 - 10mM CaCl2 - 10ul/mL Protease inhibitor from GE Healthcare   Heating sample for 5 min at 100ºC.     7) Amount of protein loaded 30ug   8) Electrophoresis/Gel conditions (Reducing or Non-reducing gel, % of the gel etc.) 15% gel Running at 100 V for 1h30min Sample buffer (4x): - 250mM Tris-HCl 0,5M pH:6,8 - 8% SDS - 20% B-mercaptoethanol - 0,012g bromophenol blue -100 mL milli-Q water - 30% glycerol     9) Transfer and blocking conditions (Buffer/time period, Blocking agent etc.) Transfer: 1h30min at 120V Transfer buffer: 9,7g Tris, 57,6g glycine, 3,2L distilled water and 0,8L methanol Transfer membranes tested: 0,45µm Hybond membrane from Amersham and 0,22µm membrane from Millipore.   Blocking conditions tested: 5% non-fat Milk diluted in TBS-T for 1h at room temperature under agitation. 5% non-fat Milk diluted in TBS-T for 2h at room temperature under agitation. 10% non-fat Milk diluted in TBS-T for 1h at room temperature under agitation. 10% non-fat Milk diluted in TBS-T for 2h at room temperature under agitation.   10) Primary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, Wash step) 1. Human Beta Defensin-1:  ab14425 (antibody) + ab50048 (rec protein) Tested dilutions: 1:500 and 1:250 for the antibody and 200 ng for the recombinant protein   2. Human Beta Defensin-2: ab66072 (antibody) + ab9872 (rec protein) Tested dilutions: 1:1000 and 1:500 for the antibody and 200 ng for the recombinant protein   3.  Human Beta Defensin-3: ab19270 (antibody) + ab50059 (rec protein) Tested dilutions: 1:1000 and 1:500 for the antibody and 200 ng for the recombinant protein   3.  Human Beta Defensin-4: ab14419 (antibody) + ab69499 (rec protein) Tested dilutions: 1:500 and 1:250 for the antibody and 200 ng for the recombinant protein   All antibodies were diluted in TBST + 5% milk and incubated overnight at 4°C under agitation. We also tried to dilute them in TBST + 5% BSA.   Wash step: 3x 5 min in TBST   11) Secondary Antibody (Manufacturer/Species/Diluent/Dilution/Incubation time, Wash step) Goat anti-mouse IgG HRP for ab14425, ab66072 and ab14419. Dilution: 1:5000 diluted in TBST +5% milk Incubation: 1 h   Goat anti-rabbit IgG HRP F(ab’) fragment for ab19270. Dilution: 1:5000 diluted in TBST +5% milk Incubation: 1 h   Wash step: 3x 5 min in TBST   12) Detection method (ECL, ECLPlus etc.) ECL Plus - Amersham   13) How many times have you tried the Western? 20 times   14) Do you obtain the same results every time? e.g. are the background bands always in the same place? The results we have are always the same for the 4 proteins. The respective bands of recombinant protein and sample do not appear. The running and transfer conditions are ok, because we have positive results with beta actin antibody in the same membranes. Also, the secondary antibody is working fine with beta actin.   15) What steps have you altered to try and optimize the use of this antibody? Primary antibody dilution, transfer membranes, blocking conditions...      

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Answer

Thank you for contacting Abcam Technical Team and for taking the time to provide some useful details of the experiments. I am very sorry to hear that your customer is are having problems with 8 of our products.  Special thanks for collecting the troubled customer's data/results in such an organized way. It may well be that the problem is either shipment/storage or protocol-related. Though you have kindly provided some details, it would be much appreciated if I could get some more information which would help me identify the source of the problem.   1) Arrival dates: Could you please confirm the exact dates when the customer received these items: - ab14425, ab50048, ab66072, ab9872, ab19270, ab50059, ab14419, ab69499 2) Orders and shipment: - Could you provide me the Abcam Order Numbers for these products? - Were these items shipped in the same package or not? 3) Marker bands: - What MW markers (i.e. range of the proteins for MW) were used for WB? I am particularly interested in the markers at the lower weight range. 4) Image: - Could you please attach an image representing the samples and the loading control on the same gels? 5) Secondary antibodies: - Has the customer used the respective secondary antibodies successfully with other primary antibodies? Could you please check if the problem does not come from the 2ndaries?   Thank you for your understanding and co-operation in this matter. I look forward to hearing from you and hope to solve this problem as soon as possible.

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Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Human Tissue sections (Pancreas)
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: DAKO antigen retrieval buffer pH9
Permeabilization
No
Specification
Pancreas
Fixative
Formaldehyde

Dr. Ilka Werner-Martini

Verified customer

Submitted Oct 12 2010

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

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