Anti-PON1 antibody [17A12] (ab24261)

Reviews (1)Q&A (6)References (19)

Overview

  • Product name

    Anti-PON1 antibody [17A12]
    See all PON1 primary antibodies

  • Description

    Mouse monoclonal [17A12] to PON1

  • Host species

    Mouse

  • Tested applications

    Suitable for: ICC/IF, IHC-P, IHC-FoFr, WB, Flow Cytmore details

  • Species reactivity

    Reacts with: Mouse, Rat, Hamster, Human, Rhesus monkey
    Predicted to work with: Rabbit

  • Immunogen

    Recombinant full length protein (His-tag) corresponding to Human PON1. His tagged recombinant Human PON1 protein purified E.coli.

  • Positive control

    • WB: HeLa, A431, NIH3T3, PC12, and HepG2 cell lysates. IHC-P: Human liver tissue. Flow Cyt: HepG2 cells. ICC/IF: HeLa cells.

  • General notes

    This product was changed from ascites to tissue culture supernatant on 18th September 2017. Lot numbers higher than GR202765 will be from tissue culture supernatant. Please note that the dilutions may need to be adjusted accordingly.

Applications

Our Abpromise guarantee covers the use of ab24261 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 at an assay dependent concentration.
IHC-P Use at an assay dependent concentration.
IHC-FoFr Use at an assay dependent concentration. PubMed: 19439227
WB Use a concentration of 2 µg/ml. Predicted molecular weight: 40 kDa.
Flow Cyt Use at an assay dependent concentration.

ab170190 - Mouse monoclonal IgG1, is suitable for use as an isotype control with this antibody.

Target

  • Function

    Hydrolyzes the toxic metabolites of a variety of organophosphorus insecticides. Capable of hydrolyzing a broad spectrum of organophosphate substrates and lactones, and a number of aromatic carboxylic acid esters. Mediates an enzymatic protection of low density lipoproteins against oxidative modification and the consequent series of events leading to atheroma formation.

  • Tissue specificity

    Plasma, associated with HDL (at protein level). Expressed in liver, but not in heart, brain, placenta, lung, skeletal muscle, kidney or pancreas.

  • Involvement in disease

    Genetic variation in PON1 is associated with susceptibility to microvascular complications of diabetes type 5 (MVCD5) [MIM:612633]. These are pathological conditions that develop in numerous tissues and organs as a consequence of diabetes mellitus. They include diabetic retinopathy, diabetic nephropathy leading to end-stage renal disease, and diabetic neuropathy. Diabetic retinopathy remains the major cause of new-onset blindness among diabetic adults. It is characterized by vascular permeability and increased tissue ischemia and angiogenesis. Note=Homozygosity for the Leu-54 allele is strongly associated with the development of retinal disease in diabetic patients.

  • Sequence similarities

    Belongs to the paraoxonase family.

  • Post-translational
    modifications

    Glycosylated.
    The signal sequence is not cleaved.
    Present in two forms, form B contains a disulfide bond, form A does not.

  • Cellular localization

    Secreted > extracellular space.
  • Information by UniProt

  • Database links

    see all

  • Alternative names

    • A esterase 1 antibody
    • A-esterase 1 antibody
    • Aromatic esterase 1 antibody
    • Arylesterase 1 antibody
    • Arylesterase B type antibody
    • ESA antibody
    • Esterase A antibody
    • K 45 antibody
    • K-45 antibody
    • MVCD5 antibody
    • Paraoxonase 1 antibody
    • Paraoxonase antibody
    • Paraoxonase B type antibody
    • Paraoxonase, plasma antibody
    • Paraoxonase1 antibody
    • PON 1 antibody
    • PON antibody
    • PON1 antibody
    • PON1_HUMAN antibody
    • Serum aryldiakylphosphatase antibody
    • Serum aryldialkylphosphatase 1 antibody
    • Serum paraoxonase/arylesterase 1 antibody
    see all

Images

  • Western blot - Anti-PON1 antibody [17A12] (ab24261)
    Western blot - Anti-PON1 antibody [17A12] (ab24261)
    All lanes : Anti-PON1 antibody [17A12] (ab24261) at 2 µg/ml

    Lane 1 : HeLa cell lysate
    Lane 2 : A431 cell lysate
    Lane 3 : NIH3T3 cell lysate
    Lane 4 : PC12 cell lysate

    Secondary
    All lanes : HRP-conjugated goat anti-mouse IgG1 at 1/5000 dilution

    Predicted band size: 40 kDa


    Exposure time: 1 minute


    Incubated with the primary overnight at 4°C. Incubated with the secondary antibody for 1 hour at room temperature.

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PON1 antibody [17A12] (ab24261)
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PON1 antibody [17A12] (ab24261)
    ab24261 (1µg/ml) staining PON1 in human liver using an automated system (DAKO Autostainer Plus). Using this protocol there is strong cytoplasmic staining of hepatocytes.
    Sections were rehydrated and antigen retrieved with the Dako 3 in 1 AR buffer citrate pH 6.0 in a DAKO PT link. Slides were peroxidase blocked in 3% H2O2 in methanol for 10 mins. They were then blocked with Dako Protein block for 10 minutes (containing casein 0.25% in PBS) then incubated with primary antibody for 20 min and detected with Dako Envision Flex amplification kit for 30 minutes. Colorimetric detection was completed with Diaminobenzidine for 5 minutes. Slides were counterstained with Haematoxylin and coverslipped under DePeX. Please note that, for manual staining, optimization of primary antibody concentration and incubation time is recommended. Signal amplification may be required.
  • Flow Cytometry - Anti-PON1 antibody [17A12] (ab24261)
    Flow Cytometry - Anti-PON1 antibody [17A12] (ab24261)

    Overlay histogram showing HepG2 cells stained with ab24261 (red line). The cells were fixed with 4% paraformaldehyde (10 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab24261, 1µg/1x106 cells) for 30 min at 22°C. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (ab91353, 2µg/1x106 cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive signal in HepG2 cells fixed with 100% methanol (5 min)/permeabilized in 0.1% PBS-Tween used under the same conditions.

  • Immunocytochemistry/ Immunofluorescence - Anti-PON1 antibody [17A12] (ab24261)
    Immunocytochemistry/ Immunofluorescence - Anti-PON1 antibody [17A12] (ab24261)

    ICC/IF image of ab24261 stained HeLa cells. The cells were 4% PFA 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 (ab24261, 1µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-mouse IgG (H+L) used at a 1/1000 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.

  • Western blot - Anti-PON1 antibody [17A12] (ab24261)
    Western blot - Anti-PON1 antibody [17A12] (ab24261)
    Anti-PON1 antibody [17A12] (ab24261) at 1/500 dilution + Recombinant Human PON1 protein (ab53376) at 0.01 µg

    Secondary
    Goat Anti-Mouse IgG H&L (HRP) preadsorbed (ab97040) at 1/500 dilution

    Developed using the ECL technique.

    Performed under reducing conditions.

    Predicted band size: 40 kDa


    Exposure time: 1 minute

References

This product has been referenced in:

  • Schilcher I  et al. Endothelial lipase increases antioxidative capacity of high-density lipoprotein. Biochim Biophys Acta Mol Cell Biol Lipids 1864:1363-1374 (2019). Read more (PubMed: 31220617) »
  • Cao J  et al. Protein markers of dysfunctional HDL in scavenger receptor class B type I deficient mice. J Transl Med 16:155 (2018). WB ; Mouse . Read more (PubMed: 29879989) »
See all 19 Publications for this product

Publishing research using ab24261? Please let us know so that we can cite the reference in this datasheet.

Customer reviews and Q&As

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1-7 of 7 Abreviews or Q&A

Western blot abreview for Anti-PON1 antibody [17A12]

 Excellent
Abreviews
abreview image
Application
Western blot
Sample
Hamster Tissue lysate - whole (Hamster (Mesocricetus auratus) liver)
Gel Running Conditions
Reduced Denaturing (10% SDS-PAGE)
Loading amount
60 µg
Treatment
Western diet (N normal, HL hyperlipemic), 16 weeks
Specification
Hamster (Mesocricetus auratus) liver
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 22°C
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Dr. Anca Sima

Verified customer

Submitted May 21 2015

Question

It will be Elisa and PON is antibody , i.e. ab24261

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Answer

Thank you for contacting us.

I can confirm that antibody ab24261 can be used in ELISA (Indirect) with human cell lysates/ proteins.

I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

Use our products? Submit an Abreview. Earn rewards!
https://www.abcam.com/abreviews

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Question

What is the concentration of HEPES in the buffer?

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Answer

Thank you for your phone call yesterday, and I apologize that we did not already have this information. I have heard back from the lab, and the buffer of ab24261 contains 10 mM HEPES. I hope this information is helpful, but please let me know if you have any further questions.

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Question

I'd like to detect human PON1 protein expressed in the liver. But PON2 and PON3 are also expressed in the liver, so I need to use a PON1 specific antibody that don't cross-react to PON2 or PON3. Do you habe any information on the cross-reactibity of your 6 anti-PON1 antibodies with PON2 and PON3? Which product do you recommend for my purpose?　Thank you.

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Answer

Sorry for the long wait. I have another update about one of the products you previously enquired. ab26930: We have not tested this product for cross reactivity with PON2 and PON3. ab24261: There is no reply from the originator and I would assume that they too have not tested the product for cross reactivity with PON2 and PON3. I am sorry I can't be of much help in this occasion but I hope that the information for ab92466 was sufficient for you to use the product in your research. If there is anything else that I can help you with, please do not hesitate to contact me.

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Question

BATCH NUMBER -- NOT SPECIFIED -- ORDER NUMBER -- NOT SPECIFIED -- DESCRIPTION OF THE PROBLEM No signal or weak signal SAMPLE tissue extract PRIMARY ANTIBODY abcam/mouse monoclonal/TBS/1:500 and 1:1000/1 Hour / overnight / 3 washes with TBST for 15 minutes . DETECTION METHOD ECL POSITIVE AND NEGATIVE CONTROLS USED Positive control (Heela cells, plasma) SAMPLE PREPARATION 50 mM phosphate buffer saline pH 7.4/ heating for 3 minutes at 100C AMOUNT OF PROTEIN LOADED 60 ug ELECTROPHORESIS/GEL CONDITIONS SDS-PAGE/12% reducing gel TRANSFER AND BLOCKING CONDITIONS transfer buffer(tris, boric acid, EDTA)/3hrs. blocking agent (5% Non-fat milk/5% BSA) SECONDARY ANTIBODY [another company] / horse / TBS / 1:2000/ 1 hour / 3 washes with TBST for 15 minutes. HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 6 HAVE YOU RUN A "NO PRIMARY" CONTROL? Yes DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes WHAT STEPS HAVE YOU ALTERED? transfer time (1 hr. and 3HR.)/anti body incubation time / anti body dilution / chnging blocking agent (BSA and non- fat milk) ADDITIONAL NOTES we did not get any band in positive control (Heela cell , plasma) as well

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Answer

I'm sorry to hear you are experiencing poor results with ab24261 in western blotting. Your protocol seems good in general and I have only a few suggestions which I help with resolve the problem: -As you are using a good positive control of HeLa cell lysate the problem does not seem to be low expression levels in this sample, however it would be worth trying a different lysis buffer as currently the buffer you use does not seem to be suitable for optimal extraction of the protein. I would therefore recommend using 20 mM Tris-HCl, pH 7.5, 1 mM EGTA and adding protease inhibitors to the buffer. Following lysis and centrifugation I would recommend to add reducing loading buffer to the supernatant and boiling for 5 min at 100C. -I am not familiar with the use of boric acid and EDTA in transfer buffers. We typically use a Tris transfer buffer made with 20% methanol and I would recommend to try this type of buffer. -it is good that you tried different blocking agents. Can I please make sure that you have tried blocking 1 hour only and incubating the antibody overnight in TBST only as sometimes the antibody binds the blocking agent rather than the membrane, if incubated too long together. -it would be worth checking that your transfer is working adequately by doing a Ponceau staining. I hope the suggestions above will help you. Please do not hesitate to contact me if those changes do not give you a better signal and I would be happy to offer you a replacement vial or refund if the antibody was purchased in the last 120 days.

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Question

I already purchased PON1 antibody. I would like to know it is rasied against what peptide sequence.

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Answer

Thank you for your enquiry. This product has been raised against the full length recombinant form of the target. I hope that this information helps.

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Question

It says that the antibody cross reacts with Mouse PON1. What is the epitope that the antibody recognizes, and how similar is the detection, ie: will 5?g of hPON1 give the same response with chemiluminescence as mPON1?

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Answer

Thank you for your enquiry. The antibody ab2461 has been tested in human samples of HeLa cell lysate and HepG2 cell lysate and in mouse samples on mouse liver tissue lysate on different gels therefore we cannot compare the amount of PON1 protein detected in those samples. In our hands the antibody works very well for both species, I hope this information will help you, please do not hesitate to contact me again if I can be of further assistance,

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