製品の概要

  • 製品名
  • 製品の詳細
    Goat polyclonal to DNAJB9
  • 由来種
    Goat
  • アプリケーション
    適用あり: ELISA, WB, IHC-Pmore details
  • 種交差性
    交差種: Human
    交差が予測される動物種: Mouse, Rat, Cow, Dog, Xenopus laevis
  • 免疫原

    Synthetic peptide:

    RGNMVTTYTDCSGQ

    , corresponding to C terminal amino acids 210-223 of Human MDG1.

  • ポジティブ・コントロール
    • Human kidney
  • 特記事項
    GenBank Accession Number – NP_036460.

製品の特性

  • 製品の状態
    Liquid
  • 保存方法
    Shipped at 4°C. Upon delivery aliquot and store at -20°C or -80°C. Avoid repeated freeze / thaw cycles.
  • バッファー
    pH: 7.30
    Preservative: 0.02% Sodium azide
    Constituents: Tris buffered saline, 0.5% BSA
  • Concentration information loading...
  • 精製度
    Immunogen affinity purified
  • 特記事項(精製)
    Purified from goat serum by ammonium sulphate precipitation followed by antigen affinity chromatography using the immunizing peptide.
  • ポリ/モノ
    ポリクローナル
  • アイソタイプ
    IgG
  • 研究分野

アプリケーション

Our Abpromise guarantee covers the use of ab6053 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 Abreviews 特記事項
ELISA 1/4000. This antibody gave a positive result in ELISA against the immunizing peptide.
WB Use at an assay dependent dilution. Predicted molecular weight: 26 kDa. Preliminary experiments gave no signal but low background in A459 and Human Skeletal Muscle extracts at up to 1µg/ml.
IHC-P Use a concentration of 4 - 6 µg/ml. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

ターゲット情報

画像

  • ab6053 at 4 µg/ml staining MDG1 in Human kidney by Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections). Steamed antigen retrieval with citrate buffer pH 6 was performed. Detected with HRP-staining.

参考文献

ab6053 has not yet been referenced specifically in any publications.

レビューと Q&A

Question

BATCH NUMBER 233128 ORDER NUMBER prof3838 DESCRIPTION OF THE PROBLEM We could’t see the expected band ( MDG1 : 26 kDa ) there was only backround. SAMPLE We have used lysates from 4 different cell lines: 293, Hela, NHI3T3 and HUVEC ( Human Ublilical Vein Endothelial Cells ) for enogenous protein. We have also tested lysates from overexpressed transfected MDG1 protein at HUVEC (this GFP-MDG1 construct we has been checked using anti-GFP antibody ). PRIMARY ANTIBODY Anti-MDG1 goat polyclonal antibody diluted 1:500 in 5% non-fat milk for 1hr. Wash 3times X 10min with western buffer ( 10% Tris 7.2 1M, 1% tween 20, 30% NaCl 5N and dd H20) DETECTION METHOD ECL Western Blotting detection system The exposure times were from 30sec – 40min POSITIVE AND NEGATIVE CONTROLS USED For the endogenous MDG1 we didn’t have a control. For the overexpressed MDG1 we used not tranfected cells for negative control. ANTIBODY STORAGE CONDITIONS Aliquot and storred at -80 C. SAMPLE PREPARATION Take lysates with lysis buffer: PBS and 1%SDS+PMSF. Sonicate lysates 2times X 10sec. Boil for 10min. Centifuge for 20min at 1200rpm and take the sup. Measure protein consentation with BSA assay. Prepare sample+Laemmli Buffer. Boil sample for 5min before loading. AMOUNT OF PROTEIN LOADED Load 40µg of protein. ELECTROPHORESIS/GEL CONDITIONS Run SDS page with 12% gel. TRANSFER AND BLOCKING CONDITIONS We blocked with non-fat milk (5% final concentration) for ½ hr at room-temperature (RT). SECONDARY ANTIBODY Anti-goat HRP diluted 1:2000 in 5% non-fat milk from [another company]. For 45min. HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 3 HAVE YOU RUN A "NO PRIMARY" CONTROL? Yes DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes WHAT STEPS HAVE YOU ALTERED? Time of exposure to primary antibody and different antibody dilutions. ADDITIONAL NOTES At the same time using the same lysates and with the same conditions we tested an other new antibody which worked. The antibody arrived thaw.

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Answer

Thank you for contacting us for technical support. I'm sorry to hear that ab6053 is not working well for you. This antibody is a FastTrack product and as such has not been fully characterised yet by our laboratory as we do not have good positive controls. We therefore currently do not guarantee this antibody to work and as such I am unable to provide you with a replacement vial. However I hope the recommendations below will help: -use a lower concentration of TBST, as a hight tris or salt concentration can prevent the antibody from binding and cause high background (typically 25 mM Tris-HCl, pH 8.0 125 mM NaCl, 0.1% Tween 20) -You mention "Take lysates with lysis buffer: PBS and 1%SDS+PMSF. Sonicate lysates 2times X 10sec. Boil for 10min. Centifuge for 20min at 1200rpm and take the sup. Measure protein concentration with BSA assay." I recommend to extract the protein RIPA or NP40 buffer containing a cocktail of protease inhibitors. As stress in known to induce its translocation to the nucleus it is possible that currently the protein is not extracted optimally. It is best to leave to gently rotate in the lysis buffer for 2 hours at 4C, then centrifugating at 4C and taking the supernatant, then measuring the protein concentration and finally to boil in reducing loading buffer. -Try blocking the membrane in 5% BSA for 1 hour, we find that it gives much less background that milk. -To maximise the correct band incubate the antibody overnight at 4C in TBST only. -I would also recommend to incubate the secondary in TBST only. Please be reassured that it is normal that the antibody arrived thawed, we pack our antibodies with ice block to retain freshness and for precaution but studies in the laboratory have shown that antibodies are stable at room temperature. I hope the above recommendations will help you.

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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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