Overview

  • Product name
    Anti-PABP antibody [10E10]
    See all PABP primary antibodies
  • Description
    Mouse monoclonal [10E10] to PABP
  • Host species
    Mouse
  • Tested applications
    Suitable for: ELISA, WB, IP, ICC/IF, Flow Cytmore details
  • Species reactivity
    Reacts with: Rabbit, Chicken, Human, Xenopus laevis
    Does not react with: Mouse, Drosophila melanogaster
  • Immunogen

    Recombinant PABP (Human) expressed from its 1.85 kbp cDNA, NcoI to SspI.

Properties

Applications

Our Abpromise guarantee covers the use of ab6125 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 Use at an assay dependent concentration.
WB Use at an assay dependent concentration. Detects a band of approximately 70 kDa (predicted molecular weight: 71 kDa).
IP Use at an assay dependent concentration.
ICC/IF Use at an assay dependent concentration. PubMed: 17977970
Flow Cyt Use 1µg for 106 cells.

ab170192 - Mouse monoclonal IgG2b, is suitable for use as an isotype control with this antibody.

 

Target

  • Relevance
    The poly(A)-binding protein (PABP), which is found complexed to the 3-prime poly(A) tail of eukaryotic mRNA, is required for poly(A) shortening and translation initiation. Grange et al. (1987) isolated a melanoma cell cDNA encoding human PABP. The predicted 633-amino acid protein contains 4 repeats of an approximately 80-amino acid unit in its N-terminal half. The authors found that this repeat region is highly conserved between human and yeast PABP and is sufficient for poly(A) binding. In vitro translation of the human PABP cDNA yielded a protein with an apparent molecular mass of 73 kD by SDS-PAGE. Northern blot analysis indicated that PABP is expressed as a 2.9-kb mRNA in human melanoma cells. Gorlach et al. (1994) noted that each of the 4 repeats of PABP is a ribonucleoprotein (RNP) consensus sequence RNA-binding domain. They determined that PABP has a pI of approximately 10.3 and is a very abundant, stable protein. Immunofluorescence studies of mammalian cells indicated that PABP is located exclusively in the cytoplasm. However, using both indirect immunofluorescence and tagging of PABP1 by fusion to the green fluorescent protein (GFP), Afonina et al. (1998) demonstrated that PABP1 shuttles between the nucleus and cytoplasm. PABP1 accumulated in the nucleus when transcription was inhibited, suggesting that active transcription is required for nuclear export of PABP1.
  • Cellular localization
    Cytoplasmic. Shuttles between the cytoplasm and the nucleus.
  • Database links
  • Alternative names
    • PAB 1 antibody
    • PAB1 antibody
    • PABP 1 antibody
    • PABP1 antibody
    • PABPC 1 antibody
    • PABPC1 antibody
    • PABPC2 antibody
    • PABPL1 antibody
    • Poly A binding protein 1 antibody
    • Poly A binding protein cytoplasmic 1 antibody
    • poly(A) binding protein, cytoplasmic 1 antibody
    • poly(A) binding protein, cytoplasmic 2 antibody
    • Polyadenylate binding protein 1 antibody
    see all

Images

  • All lanes : Anti-PABP antibody [10E10] (ab6125) at 1/2000 dilution

    Lane 1 : 10ug of protein from MCF10A cells transfected with negative control siRNA.
    Lane 2 : 10ug of protein from MCF10A cells transfected with PABP specific siRNA.
    Lane 3 : 10 ug of protein from MCF10A cells transfected with PABP specific siRNA.

    Secondary
    All lanes : Goat anti-mouse 1/10000

    Predicted band size: 71 kDa
    Observed band size: 70 kDa
    why is the actual band size different from the predicted?



    The cells were lysed with NP40 buffer with protease inhibitor cocktail, 10 micrograms of protein were separated by SDS-PAGE and transferred to PVDF membrane, blocked and blotted for two hours with PABP antibody in TBST, washed three times, secondary antibody for 1 hr (goat anti-mouse, Amersham, 1:10000).
  • PABP was immunoprecipitated using 0.5mg Hela whole cell extract, 5µg of Mouse monoclonal to PABP (ab6125) and 50µl of protein G magnetic beads (+). No antibody was added to the control (-).
    The antibody was incubated under agitation with Protein G beads for 10min, Hela whole cell extract lysate diluted in RIPA buffer was added to each sample and incubated for a further 10min under agitation.
    Proteins were eluted by addition of 40µl SDS loading buffer and incubated for 10min at 70oC; 10µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab6125.
    Secondary: Goat polyclonal to mouse IgG light chain specific (HRP) at 1/5000 dilution.
    Band: 76kDa: PABP; 25kDa.
  • ICC/IF image of ab6125 stained HeLa 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 (ab6125, 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.

  • Overlay histogram showing HeLa cells stained with ab6125 (red line). The cells were fixed with 100% methanol (5 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 (ab6125, 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 IgG2b [PLPV219] (ab91366, 2µg/1x106 cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive signal in HeLa cells fixed with 4% paraformaldehyde (10 min)/permeabilized in 0.1% PBS-Tween used under the same conditions.

References

This product has been referenced in:
  • Yang X  et al. Picornavirus 2A protease regulates stress granule formation to facilitate viral translation. PLoS Pathog 14:e1006901 (2018). Read more (PubMed: 29415027) »
  • Haque N  et al. ZFR coordinates crosstalk between RNA decay and transcription in innate immunity. Nat Commun 9:1145 (2018). Read more (PubMed: 29559679) »
See all 25 Publications for this product

Customer reviews and Q&As

1-10 of 10 Abreviews or Q&A

Answer

Thank you for contacting us.

There is not a specific concentration recommended for the use of this antibody in ELISA. Optimisation is required with different dilutions in order to assess the best one for your particular conditions. We can however suggest a good starting point to begin with, for purified antibodies tested in ELISA this corresponds to 0.1ug/ml.

Regarding the suitability of ab23710 to be used as standard with the antibody, I am afraid I cannot assure the antibody will recognize it, as its epitope has not been mapped, and we have not tested this pair in house.

However, the peptide ab23710 has been used to raise another Anti-PABP antibody:

https://www.abcam.com/pabp-antibody-ab21060.html

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

Read More

Answer

Thank you again for your calls and for your patience while we have looked into your enquiry. I apologize again for the delay in getting this information for you.

My contact at the lab confirms that this antibody has an IgG2b isotype. I've looked online and there are other antibodies with the 10E10 clone number (though one is to a different protein target, Raptor). I'm not sure how to explain the discrepancy with the PABP antibody listed as an IgG1 isotype from other vendors, though most companies seem tolist this as an IgG2b clone.

I am sorry that we don'thavemore of an explain regarding this discrepancy, but if you have any further questions or if there is anything else that we can do for you, please let me know and I'll be happy to help.

Read More

Answer

Thank you for your calls last week and earlier this week, and I apologize for the delay getting this information for you.

I've called over to the lab, andmy colleaguewho has more information about ab6125 has been out of the office. We should have some answers early next week.

I apologize again that this is taking so long, and we appreciate your patience. Please let me know if you have any questions or need anything else in the meantime.

Read More

Answer

Thank you for your enquiry. I have received information from our laboratory. The purification steps are not done aseptically (it's impossible) and so whilst I guess there could be some bacterially derived endonuclease, I think it unlikely as all our columns are stored in ethanol and all the tubing etc washed extensively before each run. Moreover, even if there were any endonucleases present, I would be surprised if they bound non specifically to protein G and so I wouldn't expect to see them in the end product. This is substantiated by the fact that we don't see any low (12-14Kd) MW bands in the preps when we run SDS-PAGE gels. Whilst we filter sterilize the preps prior to despatch, I guess if the antibody has subsequently been handled non-aseptically at any point, this could introduce bacteria which could produce endonuclease, however I think it unlikely. I hope this information will be useful to you. Should you require any further information, please do not hesitate to contact me.

Read More
Application
Immunocytochemistry/ Immunofluorescence
Sample
Human Cell (Human)
Specification
Human
Fixative
Formaldehyde
Permeabilization
Yes - 0.5 NP40
Blocking step
Serum as blocking agent for 30 minute(s) · Concentration: 10% · Temperature: 20°C

Abcam user community

Verified customer

Submitted Jul 27 2007

Question
Answer

Thank you for your phone call and I apologize for the shortage that you received. I have arranged for a free of charge replacement vial of ab6125 to be sent to your attention. It is on order# 103119 and you should receive it Tuesday. Please let me know if you have any additional questions or concerns.

Read More
Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
Human Cell lysate - whole cell (Breast epithelial cancer cell lines)
Specification
Breast epithelial cancer cell lines
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5%

Ms. Michelle Badura

Verified customer

Submitted Sep 02 2005

Answer

Thank you for your phone call. I apologize for the shortage that you received in your vial of ab6125 and I'm sending you a replacement vial free of charge. It is on order# 86753 and you should receive this by Wednesday. Please let me know if I can be of additional assistance.

Read More

Question
Answer

At Abcam, we have one centralized database to hold all of our product information, so that everything we know about this antibody is on this datasheet. We are unable to supply the epitope sequence as this is deemed commercially sensitive information. However it may be of help to you to view the references cited at the base of the data sheet. If you need any further information please get in touch once again.

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Answer

All the information that we have at this time is located on the online datasheet, and we unfortunately do not have information regarding the sequence of the epitope.

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