Overview

  • Product name
  • Description
    Rabbit polyclonal to Rpt1
  • Host species
    Rabbit
  • Specificity
    At optimal dilution there is no cross-reactivity with other members of the YTA family.
  • Tested applications
    Suitable for: WBmore details
  • Species reactivity
    Reacts with: Saccharomyces cerevisiae, Schizosaccharomyces pombe
  • Immunogen

    Recombinant (His-tagged) fusion protein:

    MPPKEDWEKY KAPLEDDDKK PDDDKIVPLT EGDIQVLKSY GAAPYAAKLK QTENDLKDIE ARIKEKAGVK ESDTGLAPSH LWDIMGDRQR

    LGEEHPLQVA, corresponding to amino acids 1-100 of Rpt1 (yeast).

  • General notes
    Dilute to working strength with phosphate buffered saline pH 7.2-7.4 and 1% normal goat serum (if a goat anti-rabbit IgG linker antibody is to be used).

Properties

Applications

Our Abpromise guarantee covers the use of ab22678 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
WB 1/10000 - 1/50000. Detects a band of approximately 52 kDa.

Target

  • Relevance
    Rpt1 in yeast is one of six ATPases of the 19S regulatory particle of the 26S proteasome involved in the degradation of ubiquitinated substrates; required for optimal CDC20 transcription; interacts with Rpn12p and the E3 ubiquitin-protein ligase Ubr1p. In the yeast S. cerevisiae, 12 different members of a novel gene family of putative ATPases have been identified and characterised. Due to their relationship with TBP1, a human immunodeficiency tat binding protein, they have been designated the YTA family (yeast tat binding analogues). The most outstanding feature of the YTA proteins and their relatives is that they share a region of high similarity which extends to some 300 amino acids in length.
  • Cellular localization
    Cytoplasmic and Nuclear
  • Database links
    • Alternative names
      • 26S protease regulatory subunit 7 homolog antibody
      • CIM5 antibody
      • Protein CIM5 antibody
      • TAT-binding homolog 3 antibody
      • YTA3 antibody
      see all

    Images

    • Anti-Rpt1 antibody (ab22678) at 1/25000 dilution + Purified yeast 26S proteasome preparation

      Developed using the ECL technique.

      Exposure time: 1 minute

    References

    ab22678 has not yet been referenced specifically in any publications.

    Customer reviews and Q&As

    1-2 of 2 Abreviews or Q&A

    Answer

    Please find below some information about the testing conditions:

    Western blot - Single dimension SDS-PAGE of a yeast whole cell extract and purified yeast 26S proteasome followed by Western blotting shows a major band at ˜52kDa. The antibody may commonly be used at dilutions in the range 1:10,000 to 1:50,000. At optimal dilution there is no cross-reactivity with other members of the YTA family.

    Immunoprecipitation - This antibody has not been characterised for use in immunoprecipitation.

    Species reactivity:- This antibody has been demonstrated to react with proteasomes from yeast.

    Reference: Schnall, R., Mannhaupt, G., Stucka, R., Tauer, R., Ehnle, S., Schwarzlose, C., Vetter, I. And Feldmann. Identification of a set of yeast genes coding for a novel family of putative ATPases with high similarity to constituents of the 26S protease complex. Yeast, 10, 1141-1155 (1994).

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    Question

    Our customer had some problem about these ab22678 antibody. They found the expected bands are faint and there are many vertical straight lines on membrane. We would appreciated if you could help them about the experiment. 1. Order details: a.. Batch number: Lot168293 b.. Abcam order or Purchase order number: ab22678 c.. Antibody storage conditions (temperature/reconstitution etc) 4? 2. Please describe the problem (high background, wrong band size, more bands, no band etc). Expected bands are faint and there are many vertical straight lines on membrane 3. On what material are you testing the antibody in WB? · Species: yeast · Cell extract or Nuclear extract: cell extract protein · Purified protein or Recombinant protein: purified protein (RPT1) 3. The lysate a.. How much protein was loaded: 70-100µg for total extract, 700-1000 ug for IP or GST pulldown a.. What lysis buffer was used: RIPA buffer b.. What protease inhibitors were used: aprotinin, leupeptin, Bestain, Pepstain A, E-64, AEBSF and DTT c.. What loading buffer was used: 6×sample buffer d.. Did you heat the samples: temperature and time: 100?, 5 mins 4. Electrophoresis/Gel conditions/ Transfer conditions a.. Reducing or non reducing gel: reducing gel b.. Gel percentage : 10% SDS-PAGE c.. Transfer conditions: 150 mA, 1 hr (semi-dry) 5. Blocking conditions a.. Buffer: TTBS b.. Blocking agent: milk, BSA, serum, what percentage: 5% nonfat milk+TTBS c.. Incubation time: Overnight d.. Incubation temperature: 4? 6. Primary Antibody a.. Specification (in which species was it raised against): rabbit · At what dilution(s) have you tested this antibody: 1:10000, 1:30000, 1:50000 · What dilution buffer was used: TTBS · Incubation time: 3-4 h · Incubation temperature: RT · What washing steps were done: wash membrane in 10 ml TTBS for 10 min, total 3 times 7. Secondary Antibody a.. Specification (in which species was it raised against)? goat anti rabbit conjugate HRP b.. At what dilution(s) have you tested this antibody: 1:10000 c.. Incubation time 1hr d.. Wash steps: wash membrane in 10 ml TTBS for 10 min, total 3 times e.. Do you know whether the problems you are experiencing come from the secondary? No, because the secondary can work for other primary antibodies 8. Detection method ECl, ECl+, other detection method: ECL 9. Background bands · Have you eliminated the possibility that any background bands could be due to the secondary antibody? (Run a “No primary” control): It is not possible, because the secondary antibody can work for other antibodies without these background · Is the blocking step sufficient? 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) Sample 1 (reprobe anti-GST-Rad23) Sample 2 11. Did you apply positive and negative controls along with the samples? Please specify. When reprobe another antibody, there are no these vertical straight lines 10. Optimization attempts · How many times have you tried the Western? 7-8 times/each student, two students are using this antibody · Do you obtain the same results every time e.g. are background bands always in the same place? yes · What steps have you altered? no

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    Answer

    Thank you for contacting Abcam regarding ab22678 and for taking the time to provide some details of the customer's Western blot experiments. I am very sorry to hear that he/she is having problem with this particular product. Here I am forwarding my comments/suggestions and hope this will help you customer: - As I understand from your e-mail that customer re-probed the membrane with ant-GST-Rad23. It may well be that the tagging interact with the recognition of the antibody. I can also see on the labelling from the third blot, that the customer carried out IP (Flag-IP) but this antibody has only been tested for Western blot application but not for immunoprecipitation (IP). - Multiple bands could be due to degradation of the proteins so it is very important to make sure that the freshly prepared lysis buffer is used for sample preparation. - Block the membrane for shorter time for 1 hr instead of overnight at 4oC. Too long blocking can also result in non-specific signals and fainted bands. I would like to emphasize that since this antibody was purchased in September 2006 - almost 5 months - I regret to inform you that the Abcam’s guarantee (free of charge replacement or credit note) does not apply for this purchase since we have no control how the antibody was stored. I am very sorry but I can’t be any of further assistance.

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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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