製品の概要

  • 製品名
  • 製品の詳細
    Sheep polyclonal to Cathepsin B
  • 由来種
    Sheep
  • アプリケーション
    適用あり: WB, Immunodiffusionmore details
  • 種交差性
    交差種: Human
  • 免疫原

    Human Cathepsin B purified from liver

製品の特性

アプリケーション

Our Abpromise guarantee covers the use of ab211 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
WB 1/500.
Immunodiffusion Use at an assay dependent dilution.

ターゲット情報

  • 機能
    Thiol protease which is believed to participate in intracellular degradation and turnover of proteins. Has also been implicated in tumor invasion and metastasis.
  • 配列類似性
    Belongs to the peptidase C1 family.
  • 細胞内局在
    Lysosome. Melanosome. Identified by mass spectrometry in melanosome fractions from stage I to stage IV.
  • Information by UniProt
  • 参照データベース
  • 別名
    • Amyloid precursor protein secretase antibody
    • APP secretase antibody
    • APPS antibody
    • CATB_HUMAN antibody
    • Cathepsin B heavy chain antibody
    • Cathepsin B1 antibody
    • CathepsinB antibody
    • CPSB antibody
    • CTSB antibody
    • cysteine protease antibody
    • OTTHUMP00000116009 antibody
    • OTTHUMP00000229510 antibody
    • OTTHUMP00000229511 antibody
    • OTTHUMP00000229512 antibody
    • OTTHUMP00000229514 antibody
    • OTTHUMP00000229515 antibody
    • OTTHUMP00000229516 antibody
    • Preprocathepsin B antibody
    see all

プロトコール

参考文献

ab211 has not yet been referenced specifically in any publications.

レビューと Q&A

1-3 of 3 Abreviews or Q&A

Question

1. Please describe the problem (high background, no staining etc). no staining 2. On what material are you testing the antibody in IHC? * Species? human cell lines * Cell line? oral and skin epithelial cells 3. How did you fix the samples? Paraformaldehyde 4. Did you apply antigen retrieval step? not required for cells 5. How did you block the unspecific binding sites? used a buffer containing PBS, bovine and human serum (see attached protocols) 6. Primary antibody * Specification (in which species was it raised against)? Cathepsin B (ab7670; will get batch no to you later) * At what dilution(s) have you tested this antibody? 1:20, 1:50, 1:100, 1:500 * Incubation time, wash steps (multiple short washes are more effective than fewer longer wash steps)? see attached protocols 7. Secondary antibody see attached protocols * What secondary antibody are you using? * Specification (in which species was it raised against)? * At what dilution(s) have you tested this antibody? * Incubation, wash steps? * Do you know whether the problems you are experiencing come from the secondary? not a problem since our +ve controls are working * What detection method are you using? 8. Background staining * Please provide an image of your staining n/a 9. Which detection system did you use? DAB or Texas Red/Dapi 10. Did you apply positive and negative controls along with the samples? Please specify. -ve controls are omitting ab; +ve controls are a +ve cell line wich expresses cathepsin and use of an ab we know works (beta 1 integrin) 11. Optimization attempts * How many times have you tried the IHC? 10 * Do you obtain the same results every time? Yes * What steps have you altered? incubation times, different detection kits - just about everything! WB Qs Could we get a detailed protocol from you, please? We have some general questions, the answers to which will enable us to investigate this matter as quickly as possible: 1. Please describe the problem (high background, wrong band size, more bands, no band etc). no bands 2. On what material are you testing the antibody in WB? * Species? human cell lines * Cell extract/ Nuclear extract? cell lysates * Recombinant protein? no 3. How much protein did you load? 20ug/lane * How did you prepare the lysate for the analysis (protease inhibitors etc)? cells just extracted into laemmli/sample buffer * Did you heat the samples? Yes - 95C for 5min 4. Primary Antibody * Specification (in which species was it raised against)? sheep Cathepsin B (ab211; will get batch no to you later) * At what dilution(s) have you tested this antibody? 1:100, 1:500, 1:1000 * Incubation time, wash step? 60min or 90min RT, 3 washes in PBS 5. Secondary Antibody * Specification (in which species was it raised against)? donkey anti-sheep biotinylated IgG * At what dilution(s) have you tested this antibody? 1:200 * Incubation time, wash step? 60min RT, 3 washes in PBS * Do you know whether the problems you are experiencing come from the secondary? it's not the secondary ab 6. What detection method are you using? ECL 7. Background bands n/a * Have you eliminated the possibility that any background bands could be due to the secondary antibody? (Run a "No primary" control) * Is the blocking step sufficient? (We recommend blocking the membrane by adding 20 ml of blocking buffer (5% non-fat dry milk, 0.1% Tween-20 in TBS). Incubate for 2 h at room temperature or overnight at 4°C with agitation) 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? * Please provide an image of your blot (as an e-mail attachment, a faxed image is not sufficient) Just a blank film 8. Optimization attempts * How many times have you tried the Western? 5 * Do you obtain the same results every time e.g. are background bands always in the same place? yes * What steps have you altered? everything! 9. Did you apply positive and negative controls along with the samples? Please specify. -ve controls -BSA lanes +ve controls - membrane blotted with anti-actin ab which always comes up +ve cell lysate (prostate cell line) does not come up with cathepsin B

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Answer

I'm sorry to hear you are experiencing problems with ab211 and ab7670. Ab7670 has not been tested in immunocytochemistry and I would recommend trying a different fixation method. Please try ice cold acetone for 5-10min and also add triton x100 (0.3%v/v) in the blocking buffer and dilution buffer for both primary and secondary antibodies, this should promote penetration of the antibody into the cells. Have you checked that the secondary antibody works with other primaries? I would also recommend incubating the antibody overnight and the secondary for 1-2hours. For ab211, I would recommend incubating the antibody overnight and using ECL+ rather than ECl as this is more sensitive. I hope these recommendations will help. Please do not hesitate to contact us again if you still have problems,

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Answer

This antibody is a polyclonal and would therefore recognize multiple epitopes across human cathepsin B1

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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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