RecombinantRabMAb

Recombinant Anti-Polyethylene glycol antibody [PEG-B-47] (ab51257)

Reviews (1)Q&A (9)References (45)

Key features and details

  • Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
  • Rabbit monoclonal [PEG-B-47] to Polyethylene glycol
  • Suitable for: ELISA, WB, IHC-P
  • Reacts with: Species independent

Overview

  • Product name

    Anti-Polyethylene glycol antibody [PEG-B-47]
    See all Polyethylene glycol primary antibodies

  • Description

    Rabbit monoclonal [PEG-B-47] to Polyethylene glycol

  • Host species

    Rabbit

  • Specificity

    This antibody recognizes the terminal methoxy group of the PEG molecule. This antibody does not cross react with non-specific targets in blood or serum. Both free form of PEG (unconjugated version) and conjugated PEG were tested by sandwich ELISA using ab51257; results showed that ab51257 only detects conjugated forms of PEG. To detect free form of PEG, we recommend using ab133471. ab133471 recognizes PEG backbone and that PEG is not required to be bound to other molecules.

  • Tested applications

    Suitable for: ELISA, WB, IHC-Pmore details

  • Species reactivity

    Reacts with: Species independent

  • Immunogen

    Chemical/ Small Molecule corresponding to Polyethylene glycol conjugated to keyhole limpet haemocyanin. KLH-Polyethylene glycol with a terminal methoxy group.

  • Positive control

    • IHC-P: Mouse injected with PEGylated protein kidney, muscle, liver and spleen tissue. WB: BSA-PEG.

  • General notes

    If you have any questions on our PEG products - please visit our Polyethylene glycol (PEG) FAQs page. 

    This product is a recombinant monoclonal antibody, which offers several advantages including:

    • - High batch-to-batch consistency and reproducibility
    • - Improved sensitivity and specificity
    • - Long-term security of supply
    • - Animal-free production
    For more information see here.

    Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMAb® patents.

    Reproducibility is key to advancing scientific discovery and accelerating scientists’ next breakthrough.

    Abcam is leading the way with our range of recombinant antibodies, knockout-validated antibodies and knockout cell lines, all of which support improved reproducibility.

    We are also planning to innovate the way in which we present recommended applications and species on our product datasheets, so that only applications & species that have been tested in our own labs, our suppliers or by selected trusted collaborators are covered by our Abpromise™ guarantee.

    In preparation for this, we have started to update the applications & species that this product is Abpromise guaranteed for.

    We are also updating the applications & species that this product has been “predicted to work with,” however this information is not covered by our Abpromise guarantee.

    Applications & species from publications and Abreviews that have not been tested in our own labs or in those of our suppliers are not covered by the Abpromise guarantee.

    Please check that this product meets your needs before purchasing. If you have any questions, special requirements or concerns, please send us an inquiry and/or contact our Support team ahead of purchase. Recommended alternatives for this product can be found below, as well as customer reviews and Q&As.

Properties

Applications

Our Abpromise guarantee covers the use of ab51257 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 a concentration of 0.5 - 5 µg/ml.
WB Use a concentration of 0.015 - 0.075 µg/ml.
IHC-P 1/100.

This antibody only works on the tissues when the animals are injected with a PEGylated protein.

ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody.

Target

  • Alternative names

    • PEG antibody

Images

  • Western blot - Anti-Polyethylene glycol antibody [PEG-B-47] (ab51257)
    Western blot - Anti-Polyethylene glycol antibody [PEG-B-47] (ab51257)
    Lane 1 : Anti-Polyethylene glycol antibody [PEG-B-47] (ab51257) at 1/1000 dilution (Purified)
    Lane 2 : Anti-Polyethylene glycol antibody [PEG-B-47] (ab51257) at 1/5000 dilution (Purified)

    All lanes : Bovine serum albumin (BSA) and PEG 5000 conjugated protein

    Lysates/proteins at 0.015 µg per lane.

    Secondary
    All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

    Observed band size: 280 kDa
    why is the actual band size different from the predicted?


    Exposure time: 120 seconds


    Blocking/Diluting buffer and concentration 5% NFDM/TBST

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Polyethylene glycol antibody [PEG-B-47] (ab51257)
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Polyethylene glycol antibody [PEG-B-47] (ab51257)

    Paraffin-embedded mouse kidney tissue stained for Polyethylene glycol using purified ab51257 at 1/50 dilution followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP Polymer) secondary. Counterstained with Hematoxylin. No staining on mouse kidney without PEGylated protein treatment (A), positive staining on mouse kidney with PEGylated protein treatment (B).

    Heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).

  • ELISA - Anti-Polyethylene glycol antibody [PEG-B-47] (ab51257)
    ELISA - Anti-Polyethylene glycol antibody [PEG-B-47] (ab51257)

    Comparison of sandwich ELISA using RabMAb/RabMAb (ab51257/ab51257) and MAb/RabMAb (Vendor A/ab51257) for capture/detection.

    Ab51257/ab51257*: Plate coated with 5 ug/mL of #47; 5 ug/mL of #47 used for detection (*Anti-PEG 47 biotin labeled).

    Vendor A/ ab51257*: Plates coated with 100 ug/mL of Vendor A Mouse MAb; 5 ug/mL of #47 used for detection (*Anti-PEG 47 biotin labeled).

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Polyethylene glycol antibody [PEG-B-47] (ab51257)
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Polyethylene glycol antibody [PEG-B-47] (ab51257)

    ab51257, unpurified,  staining mouse kidney at 1:10 dilution. Left panel: without animal injection with a PEGylated protein. Right panel: with animal injection with a PEGylated protein.

  • Western blot - Anti-Polyethylene glycol antibody [PEG-B-47] (ab51257)
    Western blot - Anti-Polyethylene glycol antibody [PEG-B-47] (ab51257)
    Anti-Polyethylene glycol antibody [PEG-B-47] (ab51257) at 0.015 µg/ml + BSA-PEG at 10 µg

    Secondary
    Goat Anti-Rabbit IgG, (H+L), HRP-conjugated at 1/1000 dilution


    Blocking buffer and concentration: 5% NFDM/TBST.

    Diluting buffer and concentration: 5% NFDM /TBST.

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Polyethylene glycol antibody [PEG-B-47] (ab51257)
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Polyethylene glycol antibody [PEG-B-47] (ab51257)

    ab51257, unpurified, staining mouse muscle at 1:10 dilution. Left panel: without animal injection with a PEGylated protein. Right panel: with animal injection with a PEGylated protein.

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Polyethylene glycol antibody [PEG-B-47] (ab51257)
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Polyethylene glycol antibody [PEG-B-47] (ab51257)

    ab51257, unpurified, staining mouse liver at 1:10 dilution. Left panel: without animal injection with a PEGylated protein. Right panel: with animal injection with a PEGylated protein.

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Polyethylene glycol antibody [PEG-B-47] (ab51257)
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Polyethylene glycol antibody [PEG-B-47] (ab51257)

    ab51257, unpurified, staining mouse spleen at 1:10 dilution. Left panel: without animal injection with a PEGylated protein. Right panel: with animal injection with a PEGylated protein.

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Polyethylene glycol antibody [PEG-B-47] (ab51257)
    Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Polyethylene glycol antibody [PEG-B-47] (ab51257)Eriksson et al PLoS One. 2014 Oct 8;9(10):e107762. doi: 10.1371/journal.pone.0107762. eCollection 2014 Reproduced under the Creative Commons license http://creativecommons.org/licenses/by/4.0/

    Immunohistochemistry images of anti-PEG (brown) and hematoxylin counterstaining (blue) of tumor (A1–A3 and B1–B3) and muscle tissue (A4 and B4) 30 minutes (A) and 2 h (B) post injection of Spago Pix

    Tumor tissue at both time points is shown at three different enlargements where the enlarged areas are indicated by the square boxes. Vessels (V), fibroblasts (Fb) and fat cells (Fc) are indicated with rounded lines. Note that the tumor tissue in panel B1–B3 originates from a mouse injected with a dose of 10 µmol Mn/kg, whereas the other tissues originate from animals administered 20 µmol Mn/kg.

    Paraformaldehyde fixed muscle and tumor tissues collected before injection and at 30 minutes and 2–4 h post-dose were paraffin embedded and thereafter cut (4 µm) and positioned on glass tissue slides. Prior to staining, the slides were placed in an antigen-retrieval solution using an automated pre-treatment module (PT-Link; Dako, Glostrup, Denmark). Slides were stained for PEG in an automated immunohistochemistry robot (Autostainer; Dako). PEG is detected with ab51257 at 0.6 μg/ml.

    (From Figure 6 Eriksson et al)

  • Other - Anti-Polyethylene glycol antibody [PEG-B-47] (ab51257)
    Other - Anti-Polyethylene glycol antibody [PEG-B-47] (ab51257)

    Method：Protein A sensor + Antigen (PEG 5K-BSA at 0.3µg/ml)+ antibody (ab51257 at  0.0312, 0.0625, 0.125, 0.25, 0.5, 1 µg/m).

    Results:

    Sample ID: PEG-5K-BSA

    KD (M): 2.41E-09

    kon(1/Ms): 2.67E+04

    kon Error: 6.81E+02

    kdis(1/s): 6.43E-05

    kdis Error: 2.92E-06

    Full X^2: 0.018444

    Full R^2: 0.982059

  • ELISA - Anti-Polyethylene glycol antibody [PEG-B-47] (ab51257)
    ELISA - Anti-Polyethylene glycol antibody [PEG-B-47] (ab51257)

    Comparison of 10 ug/well of activated linear (PEG5K) and branched (PEG40K) PEG using 5 ug/ml of ab51257 in Direct ELISA assay.

    Accuracy: By detecting the methoxy group of the PEG molecule itself, ab51257 is useful in measuring the pharmacokinetics of PEG-modified molecules in vivo. Data indicate that ab51257 detects various length Y-chain PEG molecules as well as single chain PEG molecules with equal affinity. ab51257 does not cross react with non-specific targets in blood or serum.

  • ELISA - Anti-Polyethylene glycol antibody [PEG-B-47] (ab51257)
    ELISA - Anti-Polyethylene glycol antibody [PEG-B-47] (ab51257)
    Comparison of ab51257 and Vendor A mouse MAb in Direct ELISA assy.

    Goat anti-rabbit IgG-AP used for anti-PEG-47 detection; goat anti-mouse IgM-AP used for Vendor A MAb detection.

    Fig 1a. Direct ELISA using 1 ug/mL of PEG-GCSF.

    Fig 1b. Direct ELISA using 1 ug/mL of PEG-IFN.

    Comparison to other anti-PEG: In both direct and sandwich ELISA assays, ab51257 shows greater affinity and accuracy than other anti-PEG antibodies when determining the concentration of PEG or PEG-modified proteins. Results were similar whether detecting PEG itself or PEG-modified targets.

  • ELISA - Anti-Polyethylene glycol antibody [PEG-B-47] (ab51257)
    ELISA - Anti-Polyethylene glycol antibody [PEG-B-47] (ab51257)
    ELISA assay using ab51257 to detect different forms of PEG. PEG40000-BSA is a 40 kDa PEG molecule attached to BSA. PEG5000-BSA is a 5 kDa linear PEG molecule attached to BSA.
  • ELISA - Anti-Polyethylene glycol antibody [PEG-B-47] (ab51257)
    ELISA - Anti-Polyethylene glycol antibody [PEG-B-47] (ab51257)

    ELISA Graph generated using purified ab51257 at 1μg/mL.

    Antigen concentration range:

    PEG5000 and PEG20000: 0.0078 - 0.5 μg/mL
    ADI-PEG: 0.0156 - 1 μg/mL
    PEG-BSA: 0.0039 - 0.25μg/mL
    PEG-IFN-α-2b0.0156 - 1 μg/mL

    Secondary antibody was an Alkaline Phosphatase-conjugated Goat Anti-Rabbit IgG(H+L) at 1/2500.

  • Anti-Polyethylene glycol antibody [PEG-B-47] (ab51257)
    Anti-Polyethylene glycol antibody [PEG-B-47] (ab51257)

Protocols

To our knowledge, customised protocols are not required for this product. Please try the standard protocols listed below and let us know how you get on.

Click here to view the general protocols

References (45)

Publishing research using ab51257? Please let us know so that we can cite the reference in this datasheet.

ab51257 has been referenced in 45 publications.

  • Wang Y  et al. Development of a Highly Specific Anti-drug Antibody Assay in Support of a Nanoparticle-based Therapeutic. AAPS J 22:81 (2020). PubMed: 32488626
  • Al-Mansoori L  et al. In vitro studies on CNGRC-CPG2 fusion proteins for ligand-directed enzyme prodrug therapy for targeted cancer therapy. Oncotarget 11:619-633 (2020). PubMed: 32110281
  • Hainfeld JF  et al. Iodine nanoparticles enhance radiotherapy of intracerebral human glioma in mice and increase efficacy of chemotherapy. Sci Rep 9:4505 (2019). PubMed: 30872755
  • Harmsen S  et al. Detection of Premalignant Gastrointestinal Lesions Using Surface-Enhanced Resonance Raman Scattering-Nanoparticle Endoscopy. ACS Nano 13:1354-1364 (2019). PubMed: 30624916
  • Kavita U  et al. A Fit-for-Purpose Method for the Detection of Human Antibodies to Surface-Exposed Components of BMS-986263, a Lipid Nanoparticle-Based Drug Product Containing a siRNA Drug Substance. AAPS J 21:92 (2019). PubMed: 31332587
View all Publications for this product

Customer reviews and Q&As

Show All Reviews Q&A
Submit a review Submit a question

1-10 of 10 Abreviews or Q&A

Immunocytochemistry/ Immunofluorescence abreview for Anti-Polyethylene glycol antibody [PEG-B-47]

 Excellent
Abreviews
abreview image
Application
Immunocytochemistry/ Immunofluorescence
Sample
Mouse Cell (Macrophages)
Permeabilization
Yes - 0.3% Triton X-100
Specification
Macrophages
Blocking step
Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 3% · Temperature: 21°C
Fixative
Paraformaldehyde
Read More

Abcam user community

Verified customer

Submitted Nov 08 2016

Question

Is there a pre-established method of antigen retrieval that is known to work well for this antibody.

Read More
Answer

In our validation of ab51257 in IHC-P, antigen retrieval was not necessary. ab51257 was validated internally with mice injected intraperitoneally using 100 ug of BSA-PEG as an antigen. Heart, kidney, liver and spleen tissue were stained 4 hours post-injection using Anti-PEG RabMAb without antigen retrieval.



However, some of the papers that have used the antibody in IHC-P did use an antigen retrieval step (one example used a head mediated retrieval on a DAKO autostainer). A list of publications that used the antibody in IHC are listed below, which may be of further assistance:



http://www.ncbi.nlm.nih.gov/pubmed/24749870?dopt=Abstract

http://www.ncbi.nlm.nih.gov/pubmed/25296030?dopt=Abstract

http://www.ncbi.nlm.nih.gov/pubmed/23665258?dopt=Abstract

http://www.ncbi.nlm.nih.gov/pubmed/23788571?dopt=Abstract

http://www.ncbi.nlm.nih.gov/pubmed/22745537?dopt=Abstract

Read More

Question

What is the difference between ab51257 and ab133471?

Read More
Answer

The antibody ab51257 is specific to the terminal of PEG molecule and is made up of rabbit IgG. In contrast, ab133471 is specific to the backbone of the PEG molecule and is made up of rabbit monoclonal IgM.

Read More

Question

What is the difference between ab51257 and ab53449?

Read More
Answer

Both antibodies ab51257 and ab53449 are the same clone (PEG-B-47). Product ab53449 is the biotinlyated version of ab51257.

Read More

Question

Can the PEG ELISA kit (ab138914) be used with both linear and branched PEG?

Read More
Answer

Yes, ab51257 is used in the kit as the capture antibody in the kit and it has been shown to detect both linear and branched PEG.

Read More

Question

Which PEG antibody do you recommend in detecting linear PEG? When detecting branched PEG?

Read More
Answer

Both ab51257 and ab53449 detect linear and branched PEG (based on our in-house ELISA results).

Read More

Question

Since this is an antibody against the methoxy group of PEG, is there a difference in its reactivity against free mPEG vs PEG coupled to a protein? Thanks!

Read More
Answer


Both free form of PEG (unconjugated version) and conjugated PEG were tested by sandwich ELISA using this anti-PEG antibody (ab51257); however, our results showed that this antibody does not detect unconjugated PEG.

If you need an antibody which detects the free form of PEG, I would recommend using an anti-PEG Backbone antibody, ab133471. This antibody recognizes PEG backbone and that PEG is not required to be bound to other molecules.

Read More

Question

Which PEG was this created with? Will it react with any PEG?

Read More
Answer

We used PEG with MW of 5K to generate ab51257. The smallest PEG molecule which our antibody can detect is PEG750. Based on customer feedback, the biggest PEG molecule detected is PEG 40K. Additionally, this product has been shown to detect both linear and branched PEG.

Read More

Question

Can you please let me know if your Epitomics anti-PEG antibodies can be used for immunohistochemistry with formalin-fixed tissues?

Read More
Answer

Thank you for contacting us and your interest in our products.

The Anti-Polyethylene glycol antibody [PEG-B-47] (catalogue number ab51257, 2061-1) has been used in IHC and is expected to be able to detect PEG if present in the tissue with IHC of formalin fixed, paraffin embedded sections.

This was performed at Epitomics with mice injected intraperitoneally using 100 ug of BSA-PEG as an antigen. Heart, kidney, liver and spleen tissue were stained 4 hours post-injection using Anti-PEG RabMAb (Cat #2061-1). Attached please find images of the results obtained.

I hope this information has been helpful. If I can be of any further assistance, please do not hesitate to let me know.

Read More

Question

Sorry to bother you . But it is urgent.

ab51257= concentration ?

Regards

Read More
Answer

Thank you for your enquiry regarding ab51257.

I have spoken to my colleagues in the Lab and they have confirmed me that the concentration is batch dependent but the actual concentration of this product is printed on the label of the vial.

If you need any further assistance, please do not hesitate to contact me.

Read More

Please note: All products are "FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES"
For licensing inquiries, please contact partnerships@abcam.com