Overview

  • Product name
    Anti-Proteasome Inhibitor subunit 1 PI3 1 antibody
    See all Proteasome Inhibitor subunit 1 PI3 1 primary antibodies
  • Description
    Goat polyclonal to Proteasome Inhibitor subunit 1 PI3 1
  • Host species
    Goat
  • Specificity
    This antibody is expected to recognise isoform 1 (NP_006805, NP_848693) only, not isoform 2 (NP_848694).
  • Tested applications
    Suitable for: ELISA, WBmore details
  • Species reactivity
    Reacts with: Human
    Predicted to work with: Mouse, Rat
  • Immunogen

    Synthetic peptide: DHLPPPGYDDMYL, corresponding to C terminal amino acids 259-271 of Human Proteasome Inhibitor subunit 1 PI3 1.

  • Positive control
    • Human Kidney lysate.
  • General notes
    Principal Names – PSMF1; PI31; proteasome (prosome, macropain) inhibitor subunit 1 (PI31); proteasome inhibitor hP131 subunit Official Gene Symbol - PSMF1 GenBank Accession Number – NP_006805; NP_848693 LocusLink ID - 9491 (human); 228769 (mouse).

Properties

  • Form
    Liquid
  • Storage instructions
    Shipped at 4°C. Upon delivery aliquot and store at -20°C or -80°C. Avoid repeated freeze / thaw cycles.
  • Storage buffer
    Preservative: 0.02% Sodium Azide
    Constituents: 0.5% BSA, 5mg/ml Tris, pH 7.3
  • Concentration information loading...
  • Purity
    Immunogen affinity purified
  • Purification notes
    Purified from goat serum by ammonium sulphate precipitation followed by antigen affinity chromatography using the immunizing peptide.
  • Clonality
    Polyclonal
  • Isotype
    IgG
  • Research areas

Applications

Our Abpromise guarantee covers the use of ab3893 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
WB
  • Application notes
    ELISA: 1/32000.
    WB: Use at a concentration of 1 - 3 µg/ml.


    Not yet tested in other applications.
    Optimal dilutions/concentrations should be determined by the end user.
  • Target

    Images

    • ab3893 staining (1µg/ml) of Human Kidney lysate (RIPA buffer, 30µg total protein per lane).  Primary incubated for 1 hour.  Detected by western blot using chemiluminescence.

       

      ab3893 staining (1µg/ml) of Human Kidney lysate (RIPA buffer, 30µg total protein per lane). Primary incubated for 1 hour. Detected by western blot using chemiluminescence.

    References

    ab3893 has not yet been referenced specifically in any publications.

    Customer reviews and Q&As

    1-2 of 2 Abreviews or Q&A

    Abcam has not validated the combination of species/application used in this Abreview.
    Application
    Western blot
    Sample
    Mouse Cell lysate - whole cell (MEF)
    Gel Running Conditions
    Reduced Denaturing
    Loading amount
    15 µg
    Specification
    MEF
    Blocking step
    Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C

    Abcam user community

    Verified customer

    Submitted May 17 2017

    Question

    I have already sent a picture with details showing the latest attempt to get this antibody to work. But I will try to answer these questions and send the picture again. 1. Please describe the problem (high background, wrong band size, more bands, no band etc). There doesn't appear to be a correct or distinguishable band in the area that PI31 is supposed to be. Since the control is not working, I have no idea if my samples work or not, plus there is an incredible amount of background. My blots don't give me any usable data, and I don't know if it's because that protein may not be present in my samples (it should be), or that the primary antibody or something else is not working. 2. On what material are you testing the antibody in WB? •Species? •Cell extract/ Nuclear extract? •Purified protein? •Recombinant protein? I am testing the antibody with your control (ab7920), which doesn't give me a reaction. Then my samples are rat skeletal muscle extracts (homogenized tissue). I have also tried, kidney lysates and HeLa cell lysates with no reaction. 3. How much protein did you load? •How did you prepare the lysate for the analysis (protease inhibitors etc)? •Did you heat the samples? I have been trying to do a curve with my samples, so the load has been from 2-40 ug. Samples are not boiled, but samples are diluted in SDS reducing buffer which contains, beta-mercaptoethanol, glycerol, Tris, SDS, bromthymol blue. The human kidney control was not boiled, and arrived in SDS buffer, so not diluted either. 4. Primary Antibody •Specification (in which species was it raised against)? •At what dilution(s) have you tested this antibody? •Incubation time, wash step? I used ab3893, lot 15623, which was raised in goat. I have used the recommended dilution of 0.5-2ug/ml, incubated at room temp. for 1-2 hours. Wash was atleast 3 times for 5 minutes each. 5. Secondary Antibody •Specification (in which species was it raised against)? •At what dilution(s) have you tested this antibody? •Incubation time, wash step? •Do you know whether the problems you are experiencing come from the secondary? I used a bovine-anti-goat, and then your rabbit-anti-goat (ab6742), both are Alkaline phosphatase. I have used them at 1:3000 - 1:15,000 (normal secondary diln. for skeletal muscle is 1:5000). I have tried secondary only with several diln's as well. As the blot picture shows, the secondary only(ab6742) doesn't look good either. Again, incubation is 1 hour room temp, the same wash steps as before. 6. What detection method are you using? Alkaline phosphatase 7. Background bands •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¡ãC with agitation) •Are your washing steps sufficiently stringent? (Multiple short washes are more effective than fewer longer wash steps) •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) I have included a picture of secondary only, along with a curve of my samples and control. The bands all look the same as the primary antibody. We do not have milk in this lab for blocking or washing (our gelatin has always worked-ingredients are listed on the ppt slide). Also, secondary reaction would be way to high if it was done more than 1 hour, I am already using a 1:15,000 (a very small amount of secondary antibody) 8. Optimization attempts •How many times have you tried the Western? •Do you obtain the same results every time e.g. are background bands always in the same place? •What steps have you altered? I have tried this primary antibody 6-10 times. It pretty much always looks this bad. I have altered times for incubation, secondary antibody, different combos of primary and secondary, different "positive" controls, increasing the primary conc. 9. Did you apply positive and negative controls along with the samples? Please specify. the only positive control I am aware of that is supposed to work, is the human kidney lysates (ab7920). As far as a neg. control, I don't have any purified protein or anything else on the blots.

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    Answer

    Thank you for sending the details of your protocol and the picture of your blot. I really think that the background bands are due to the secondary. What appears to be happening is that the secondary is binding additional proteins and reacting with the blocking agent. You need to try to get rid of all the bands in the secondary only control. To do this, I suggest that you change the blocking agent. Try 5% non-fat dried milk with Tween 20 and also try 3% BSA. You may also need to try a lower concentration of the secondary and also, I suggest that you increase your number of washes and make sure that there is Tween 20 in your washes. The gelatin that you use for the blocking and washes may very well be unsuitable with this antibody. Let me know how these suggestions work out for you.

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