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Recombinant human BD-3 protein (ab50059)

Submit a review Q&A (3)

Key features and details

  • Expression system: Escherichia coli
  • Purity: >= 98% SDS-PAGE
  • Endotoxin level: < 0.100 Eu/µg
  • Active: Yes
  • Suitable for: SDS-PAGE, Functional Studies

製品の詳細

  • 製品名

    Recombinant human BD-3 protein
    BD-3 タンパク質・ペプチド 製品一覧

  • 精製度

    >= 98 % SDS-PAGE.
    Purity determined by SDS-PAGE gel and HPLC analyses.

  • エンドトキシン・レベル

    < 0.100 Eu/µg

  • 発現系

    Escherichia coli

  • タンパク質長

    Full length protein

  • Animal free

    No

  • 由来

    Recombinant

    • 生物種

      Human

    • 領域

      1 to 45

特性

Our Abpromise guarantee covers the use of ab50059 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

  • アプリケーション

    SDS-PAGE

    Functional Studies

  • 製品の状態

    Lyophilized

  • 備考

    Previously labelled as beta Defensin 3. 

  • Concentration information loading...

前処理および保存

  • 保存方法および安定性

    Shipped at 4°C. The lyophilized protein is stable for a few weeks at room temperature. Store at -20°C long term.

    This product is an active protein and may elicit a biological response in vivo, handle with caution.

  • 再構成
    Reconstitute to 1mg/ml using sterile water.

関連情報

  • 別名

    • BD 3
    • BD-3
    • BD3
    • beta 103
    • Beta defensin 103A
    • Beta defensin 103A precursor
    • Beta defensin 3
    • Beta-defensin 103
    • Beta-defensin 3
    • D103A_HUMAN
    • DEF B3
    • DEFB 103
    • DEFB 103A
    • DEFB 3
    • DEFB-3
    • DEFB103
    • DEFB103A
    • DEFB103B
    • DEFB3
    • Defensin
    • Defensin beta 103A
    • Defensin beta 3
    • Defensin like protein
    • Defensin-like protein
    • HBD 3
    • hBD-3
    • HBD3
    • HBP 3
    • HBP3
    • mBD3
    see all

  • 機能

    Exhibits antimicrobial activity against Gram-positive bacteria S.aureus and S.pyogenes, Gram-negative bacteria P.aeruginosa and E.coli and the yeast C.albicans. Kills multiresistant S.aureus and vancomycin-resistent E.faecium. No significant hemolytic activity was observed.

  • 組織特異性

    Highly expressed in skin and tonsils, and to a lesser extent in trachea, uterus, kidney, thymus, adenoid, pharynx and tongue. Low expression in salivary gland, bone marrow, colon, stomach, polyp and larynx. No expression in small intestine.

  • 配列類似性

    Belongs to the beta-defensin family.

  • 細胞内局在

    Secreted.
  • Information by UniProt

プロトコール

To our knowledge, customised protocols are not required for this product. Please try the standard protocols listed below and let us know how you get on.

Click here to view the general protocols

参考文献 (0)

ab50059 を使用した論文を発表された方は、こちらまでお知らせください。データシートに掲載させていただきます。

ab50059 は論文での使用が確認できていません。

レビューと Q&A

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Question


Customer has performed the Dot-Blotprotocolas suggested;please findthe results attached.

Regarding the storage:
- antibodies were stored at -20°C.
- recombinant proteins were reconstituted with 100 ul PBS + 15% glycerol, aliquoted andthen stored at -80°C.



Regarding the WB protocol, she said shealways do the ponceau stain to confirm the transfer, and her transfer buffer does not contain SDS.



Thank you for you support.



I am looking forward to hearing from you.

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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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Question

Dear madam/sir Thank you for your reply. I would also like to ask whether you have any information on the folding/3D strucuture  of this peptide? Thank you 

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Answer

Thank you for your reply. Unfortunately, we do not have information about the folding/3D structure of ab50059. We did not perform any testing on the specific folding structure to determine the 3D structure of this protein. However, technically, the protein should be folded correctly since it is active. I am sorry that I did not have a more detailed answer for you on this occasion and hope that this information is nevertheless helpful.

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