Overview

  • Product name
  • Description
    Rabbit polyclonal to PON3
  • Host species
    Rabbit
  • Tested applications
    Suitable for: ICC/IF, IHC-P, WBmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Human
  • Immunogen

    Synthetic peptide conjugated to KLH derived from within residues 50 - 150 of Human PON3.

    Read Abcam's proprietary immunogen policy (Peptide available as ab42321.)

  • Positive control
    • WB: Human skeletal muscle, mouse liver, rat liver and rat skeletal muscle tissue lysates. ICC/IF: HepG2 cells.

Properties

Applications

Our Abpromise guarantee covers the use of ab42322 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
ICC/IF Use a concentration of 5 µg/ml.
IHC-P Use a concentration of 1 µg/ml.
WB Use a concentration of 1 µg/ml. Detects a band of approximately 43 kDa (predicted molecular weight: 40 kDa).

Target

Images

  • ICC/IF image of ab42322 stained HepG2 cells. The cells were 4% formaldehyde fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab42322, 5µg/ml) overnight at +4°C. The secondary antibody (green) was ab96899, DyLight® 488 goat anti-rabbit IgG (H+L) used at a 1/250 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.

  • All lanes : Anti-PON3 antibody (ab42322) at 1 µg/ml

    Lane 1 : Human skeletal muscle tissue lysate - total protein (ab29330)
    Lane 2 : Liver (Mouse) Tissue Lysate - normal tissue
    Lane 3 : Liver (Rat) Tissue Lysate
    Lane 4 : Skeletal Muscle (Rat) Tissue Lysate - normal tissue (ab29376)

    Lysates/proteins at 10 µg per lane.

    Secondary
    All lanes : Goat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution

    Performed under reducing conditions.

    Predicted band size: 40 kDa
    Observed band size: 43 kDa
    why is the actual band size different from the predicted?
    Additional bands at: 49 kDa. We are unsure as to the identity of these extra bands.

  • IHC image of ab42322 staining PON3 in Human liver formalin fixed paraffin embedded tissue section, performed on a Leica BondTM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab42322, 1µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

    For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

References

This product has been referenced in:
  • Pei JF  et al. Human paraoxonase gene cluster overexpression alleviates angiotensin II-induced cardiac hypertrophy in mice. Sci China Life Sci 59:1115-1122 (2016). Read more (PubMed: 27578362) »
  • Birkus G  et al. Intracellular Activation of Tenofovir Alafenamide and the Effect of Viral and Host Protease Inhibitors. Antimicrob Agents Chemother 60:316-22 (2015). Read more (PubMed: 26503655) »
See all 3 Publications for this product

Customer reviews and Q&As

1-3 of 3 Abreviews or Q&A

Question
Answer

The PON3 antibody that I would like to send to you free-of-charge is ab108964. This is a rabbit monoclonal that has given good western blot results for a variety of human tissue and cell lysates. However, it is not suitable for IHC or ICC.

Click here (or use the following: https://www.abcam.com/index.html?datasheet=108964).

Will this be acceptable for your research?

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Answer

Thank you for your email and the additional information. This is very helpful.

I would suggest that if no protease inhibitors were used, to include them in your lysis buffer freshly, before lysing the cells (e.g. ab65621 protease Inhibitor cocktail, https://www.abcam.com/Protease-Inhibitor-Cocktail-ab65621.html). This is crucial to prevent degradation of proteins by proteases and other enzymes that are released from cell compartments during lysis.

What was the result for PON2 after 1/800 dilution. You can also use more antibody, but 1/800 is the suggested starting dilution.
For PON 3, please let me know your results as well (with 1/400).
Also, what bands did you obtain for the liver lysate?

In general, you can always increase the protein amount if the signal is weak as this can help with obtaining a band.

Is the secondary antibody working well with other primary antibodies?

Please let me know. If the results are still not satisfying, we can offer either free of charge replacements for each antibody, or a credit or refund. There will be no need to return the antibodies to us.

I look forward to hear back from you.

Use our products? Submit an Abreview. Earn rewards!
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Read More

Answer

Thank you for bringing this to our attention.

I am sorry to read that you are having difficulty obtaining a western blot signal. I may be able to make some recommendations but I will need a few more details about your samples and protocol.

Can you please tell me what kind of human tissues you are working with? How are the samples prepared? In particular, are they prepared with protease inhibitors? Are you confident that the PON proteins are expressed in these tissues?

How much of the sample do you load per lane of the gel?

Do you have evidence that the proteins transfer successfully from the gel to the membrane?

Have you stained blots of the same samples for any other proteins?

For these two antibodies, we recommend a working solution of at least 1 ug/ml of antibody. The dilution factor will depend on the lot, but for the lots you have, the dilution of ab40969 that achieves 1 ug/ml is 1/800, and for ab42322, 1/400. incubating the blots with a more concentrated solution of antibody may help, but this assumes that at least 20ug of protein is loaded per lane. More important is the quality of the sample, and a positive control. For each of these proteins, a liver lysate will serve as a positive control. We also have purified PON2, ab53379.

I look forward to your reply.

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