Overview

  • Product name
    Anti-beta Defensin 1 antibody [M11-14b-D10]
    See all beta Defensin 1 primary antibodies
  • Description
    Mouse monoclonal [M11-14b-D10] to beta Defensin 1
  • Host species
    Mouse
  • Tested applications
    Suitable for: ELISA, WB, IHC-Pmore details
  • Species reactivity
    Reacts with: Human
    Predicted to work with: Chimpanzee, Baboon, Macaque monkey
  • Immunogen

    Synthetic peptide:

    DHYNCVSSGGQCLYSACPIFTKIQGTCYRGKAKCCK

    , corresponding to amino acids 1-36 of Human beta 1 Defensin.

Properties

  • Form
    Liquid
  • Storage instructions
    Shipped at 4°C. Upon delivery aliquot and store at -20°C. Avoid freeze / thaw cycles.
  • Storage buffer
    pH: 7.40
    Constituent: 0.79% Tris HCl
  • Concentration information loading...
  • Purity
    Protein L purified
  • Clonality
    Monoclonal
  • Clone number
    M11-14b-D10
  • Isotype
    IgG1
  • Research areas

Applications

Our Abpromise guarantee covers the use of ab14425 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 beta Defensin 1 protein (ab50048).
IHC-P Use a concentration of 2 µg/ml.

Target

  • Function
    Has bactericidal activity.
  • Tissue specificity
    Plasma.
  • Sequence similarities
    Belongs to the beta-defensin family.
  • Cellular localization
    Secreted.
  • Information by UniProt
  • Database links
  • Alternative names
    • BD 1 antibody
    • BD-1 antibody
    • BD1 antibody
    • beta 1 antibody
    • Beta defensin 1 antibody
    • Beta-defensin 1 antibody
    • DEFB 1 antibody
    • DEFB1 antibody
    • DEFB1_HUMAN antibody
    • DEFB101 antibody
    • Defensin antibody
    • Defensin beta 1 antibody
    • Defensin beta 1 preproprotein antibody
    • HBD 1 antibody
    • hBD-1 antibody
    • HBD1 antibody
    • MGC51822 antibody
    see all

Images

  • ab14425 (2µg/ml) staining beta defensin in human kidney, using an automated system (DAKO Autostainer Plus). Using this protocol there is strong staining in the epithelial layers of the loops of Henle, distal tubules and collecting ducts.
    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.

References

This product has been referenced in:
  • Gazi U  et al. Tonsillar antimicrobial peptide (AMP) expression profiles of periodic fever, aphthous stomatitis, pharyngitis, cervical adenitis (PFAPA) patients. Int J Pediatr Otorhinolaryngol 110:100-104 (2018). IHC-P ; Human . Read more (PubMed: 29859567) »
  • Roth M  et al. Broncho Vaxom (OM-85) modulates rhinovirus docking proteins on human airway epithelial cells via Erk1/2 mitogen activated protein kinase and cAMP. PLoS One 12:e0188010 (2017). Read more (PubMed: 29182620) »
See all 6 Publications for this product

Customer reviews and Q&As

1-8 of 8 Abreviews or Q&A

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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Answer

Thank you for your enquiry. Human beta-1 defensin is expressed in kidney. Therefore, I would suggest you can use a human kidney whole cell lysate such as our peptide product ab30203. I have placed a link here to a reference I hope you may find useful from BMC Infectious Diseases; Expression of human beta-defensins 1 and 2 in kidneys with chronic bacterial infection (Jan Lehmann1 et al) http://www.biomedcentral.com/content/pdf/1471-2334-2-20.pdf Alternatively, you could use a purified beta-1 defensin peptide/protein. I hope this is helpful. Please do not hesitate to contact us again if you require more assistance.

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Question
Answer

Thank you for your enquiry. In testing this antibody, the plates were coated with linear and tri-cyclic ß-Defensins (1 µg/1ml) and a good signal was seen with an antibody-dilution of 0.1 µg/ml. Please find the protocol used in quality testing below. I hope this information helps, please do not hesitate to contact us if you need any more advice or information. We coated with linear and tri-cyclic ß-Defensins (1 µg/1ml) and we achieved a good signal having an antibody-dilution of 0,1 µg/ml. REAGENTS Coating buffer: 0.05 M Na2CO3 adjust pH to 9.6 with solid NaHCO3 Blocking buffer: 2% BSA in PBS Washbuffer: 1.37 Nacl, 26.8 mM KCl, 14.7mM KH2PO4, 81 mM Na2HPO4, 1 % TRITON-X-100, 0.02% THIMEROSAL First antibody: dilute in 2% BSA in PBS Second antibody: dilute in wash buffer Substrate : TMB-solution (BIOFX, USA) Stop solution: 0.4 M H2SO4 Microtiterplate NUNC Medisorb PROCEDURE • pipette 100 µl of peptide (1000 ng/ml in coating buffer) into the wells of a microtitre plate • incubate over night at 4°C • decant the contents of the plate and add 250 µl blocking buffer • incubate 1 hour at room temperature, shaking on a horizontal mixer • decant the content of the plate and wash the cavities 5 x with 250 µl of wash buffer • pipette 100 µl of antiserum (respective dilution in 2% BSA / PBS) • incubate at least 3 hrs at room temperature (or over night at 4°C), shaking on a horizontal mixer • decant the content of the plate and wash the cavities 5 x with 250 µl of wash buffer • pipette 100 µl of POX-labelled second antibody (e.g. if first antibody is rabbit: donkey anti-rabbit-POX can be used at 1: 10000 in wash buffer) • incubate 1 hr at room temperature, shaking on a horizontal mixer • decant the content of the plate and wash the cavities 5 x with 250 µl of wash buffer • pipette 100 µl TMB-solution • incubate for 15 - 30 minutes at room temperature, shaking slightly, until sufficient color differentiation has been observed • add 50 µl of stop solution and mix shortly • measure the extinction of the samples at 450 nm (reference wave length 620 nm)

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Answer

Thank you for taking the time to submit this enquiry. The Ab that you have been utilising has not been specifically tested for use in dot blot Elisa but standard direct/indirect ELISA. I would be grateful if you could provide me with details relating to the protocol that you have utilised in order for me to look into this matter in more detail. My initial concern would be that a kit that has been specifically design for Western blot analysis is being utilised as a detection system. The “Western Breeze” procedure specifically mentions not allowing the membrane to dry out; however I believe that during dot blot ELISA the antigen is spotted onto air dried membrane (pre soaked in PBS) before being dried for a further hour. Do you have a standard protocol for dot blot ELISA within your lab, as I would be inclined to optimise a published dot blot protocol rather than utilising kits optimised for use in other applications. I look forward to hearing form you shortly.

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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.

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Answer

Thank you for your enquiry. Further to correspondence with the source of this antiserum I have been informed that the antiserum was raised against tricycle human ß-Defensin 2 antigen. Unfortunately they could not provide details of whether the antisera had been tested against the native protein or your protein standards. I have therefore not been able to find evidence that these sera have been tested against the native form of this protein. I would definitely be interested to know how these sera performed against the denatured forms of your standards.

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Answer

We are sorry, but it is not known to us. It has not been tested.

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