Overview

  • Product name

    Anti-TRF1 antibody [TRF-78]
    See all TRF1 primary antibodies
  • Description

    Mouse monoclonal [TRF-78] to TRF1
  • Host species

    Mouse
  • Tested applications

    Suitable for: ELISA, WB, IHC-P, ICC/IFmore details
  • Species reactivity

    Reacts with: Human
  • Immunogen

    Human TRF1 protein produce in baculovirus

  • Positive control

    • ICC/IF: HeLa cells. WB: HeLa, HEK-293, HepG2 and U-2 OS cell lysate. HeLa nuclear cell lysate.

Properties

Applications

Our Abpromise guarantee covers the use of ab10579 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 a concentration of 2 - 4 µg/ml. Predicted molecular weight: 50 kDa.
IHC-P 1/100. PubMed: 18641004
ICC/IF 1/100.

Target

  • Function

    Binds the telomeric double-stranded TTAGGG repeat and negatively regulates telomere length. Involved in the regulation of the mitotic spindle. Component of the shelterin complex (telosome) that is involved in the regulation of telomere length and protection. Shelterin associates with arrays of double-stranded TTAGGG repeats added by telomerase and protects chromosome ends; without its protective activity, telomeres are no longer hidden from the DNA damage surveillance and chromosome ends are inappropriately processed by DNA repair pathways.
  • Tissue specificity

    Highly expressed and ubiquitous. Isoform Pin2 predominates.
  • Sequence similarities

    Contains 1 HTH myb-type DNA-binding domain.
  • Domain

    The acidic N-terminal domain binds to the ankyrin repeats of TNKS1 and TNKS2. The C-terminal domain binds microtubules.
    The TRFH dimerization region mediates the interaction with TINF2.
  • Post-translational
    modifications

    Phosphorylated preferentially on Ser-219 in an ATM-dependent manner in response to ionizing DNA damage.
    ADP-ribosylation by TNKS1 or TNKS2 diminishes its ability to bind to telomeric DNA.
    Ubiquitinated by RLIM/RNF12, leading to its degradation by the proteasome. Ubiquitinated by a SCF (SKP1-CUL1-F-box protein) ubiquitin-protein ligase complex, leading to its degradation by the proteasome.
  • Cellular localization

    Nucleus. Cytoplasm > cytoskeleton > spindle. Chromosome > telomere. Colocalizes with telomeric DNA in interphase and metaphase cells and is located at chromosome ends during metaphase. Associates with the mitotic spindle.
  • Information by UniProt
  • Database links

  • Alternative names

    • hTRF1 AS antibody
    • NIMA interacting protein 2 antibody
    • NIMA-interacting protein 2 antibody
    • PIN 2 antibody
    • PIN2 antibody
    • t TRF1 antibody
    • Telomeric protein Pin2 antibody
    • Telomeric protein Pin2/TRF1 antibody
    • Telomeric repeat binding factor (NIMA interacting) 1 antibody
    • Telomeric repeat binding factor 1 antibody
    • Telomeric repeat binding protein 1 antibody
    • Telomeric repeat-binding factor 1 antibody
    • TERF 1 antibody
    • Terf1 antibody
    • TERF1_HUMAN antibody
    • TRBF 1 antibody
    • TRBF1 antibody
    • TRF 1 antibody
    • TRF antibody
    • TTAGGG repeat binding factor 1 antibody
    • TTAGGG repeat-binding factor 1 antibody
    see all

Images

  • Immunocytochemistry/ Immunofluorescence analysis of HeLa (Human epithelial cell line from cervix adenocarcinoma) cells labeling TRF1 with ab10579. Cells were fixed and permeabilized with 4% paraformaldehyde followed by 0.5% Triton X-100. Fixed cells were stained with 10 μg/mL Anti-TRF1 antibody [TRF-78] (ab10579).  The antibody was developed using Goat Anti-Mouse IgG, Cy3 conjugate. Cells were counterstained with DAPI (blue) to stain nuclei.

  • All lanes : Anti-TRF1 antibody [TRF-78] (ab10579) at 1/1000 dilution

    Lane 1 : SAOS2 (Human osteosarcoma cell line) cell lysate
    Lane 2 : U-2 OS (Human bone osteosarcoma epithelial cell line) cell lysate

    Lysates/proteins at 20 µg per lane.

    Secondary
    All lanes : Goat anti Mouse polyclonal IRDye 800CW at 1/1000 dilution

    Predicted band size: 50 kDa


    Exposure time: 5 minutes


    Blocking step
    Milk as blocking agent for 2 hours · Concentration: 5% · Temperature: 21°C.
    Incubation time
    12 hours · Temperature: 4°C · Diluent: 5% milk in TBST.
  • All lanes : Anti-TRF1 antibody [TRF-78] (ab10579) at 4 µg/ml

    Lane 1 : HeLa (Human epithelial cell line from cervix adenocarcinoma) nuclear cell lysate
    Lane 2 : HeLa cell lysate
    Lane 3 : HEK-293T (Human epithelial cell line from embryonic kidney transformed with large T antigen) cell lysate
    Lane 4 : U-2 OS (Human bone osteosarcoma epithelial cell line) cell lysate
    Lane 5 : HepG2 (Human liver hepatocellular carcinoma cell line) cell lysate

    Secondary
    All lanes : Goat Anti-Mouse IgG-Peroxidase

    Predicted band size: 50 kDa

  • Immunofluorescent imaging of human cells (U2OS) with ab10579 confirms the specificity of this antibody.  A few intense nuclear foci are seen in interphase cells, corresponding to telomeric localisation.  The complete absence of background nuclear or cytoplasmic staining confirms the specificity of this antibody. This image is in exact agreement with numerous published reports.

    IF was performed with a standard paraformaldehyde technique (fixed in PBS buffered PFH 4% for 5 minutes, permeabilised with 0.5% triton-PBS for 5 minutes, blocked with 5% milk / 0.2% tween for one hour.  Primary antibody used at 1/100 in 5% milk / 0.2% TWEEN for one hour, secondary antibody for 30 minutes.  All blocking and incubation steps carried out at 37 degrees. Nuclei are visualised using Hoechst stain. 

  • ab10579 at 1/500 staining human HeLa (Human epithelial cell line from cervix adenocarcinoma) cells by ICC/IF. The cells were parafomaldehyde fixed and blocked with BSA prior to incubation with the antibody for 45 minutes. An Alexa Fluor® 555 conjugated donkey anti-mouse antibody was used as the secondary.

    See Abreview

References

This product has been referenced in:

  • Wallace HA  et al. Condensin II subunit NCAPH2 associates with shelterin protein TRF1 and is required for telomere stability. J Cell Physiol N/A:N/A (2019). Read more (PubMed: 31026066) »
  • Wang L  et al. Dual roles of TRF1 in tethering telomeres to the nuclear envelope and protecting them from fusion during meiosis. Cell Death Differ 25:1174-1188 (2018). Read more (PubMed: 29311622) »
See all 43 Publications for this product

Customer reviews and Q&As

1-10 of 21 Abreviews or Q&A

Application
Immunocytochemistry/ Immunofluorescence
Sample
Mouse Cell (MC3T3)
Permeabilization
Yes - NP40
Specification
MC3T3
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 3% · Temperature: 21°C
Fixative
Formaldehyde

Abcam user community

Verified customer

Submitted Mar 05 2019

Application
Western blot
Sample
Human Cell lysate - whole cell (U2OS and SAOS-2)
Gel Running Conditions
Reduced Denaturing
Loading amount
20 µg
Specification
U2OS and SAOS-2
Blocking step
Milk as blocking agent for 2 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 21°C

Abcam user community

Verified customer

Submitted Feb 19 2019

Application
Immunocytochemistry/ Immunofluorescence
Sample
Human Cell (U2OS)
Permeabilization
Yes - NP40
Specification
U2OS
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 3% · Temperature: 21°C
Fixative
Paraformaldehyde

Abcam user community

Verified customer

Submitted Feb 11 2019

Application
Immunocytochemistry/ Immunofluorescence
Sample
Mouse Cell (Brain)
Permeabilization
Yes - Triton Tx 0,3%
Specification
Brain
Blocking step
Serum as blocking agent for 2 hour(s) and 0 minute(s) · Concentration: 10% · Temperature: 23°C
Fixative
Paraformaldehyde

Dr. Milan Stojiljkovic

Verified customer

Submitted Mar 10 2016

Application
Western blot
Sample
Mouse Cell lysate - whole cell (HEK293T-TRF1 HEK293T-TRF2)
Gel Running Conditions
Reduced Denaturing
Loading amount
100 µg
Specification
HEK293T-TRF1 HEK293T-TRF2
Blocking step
(agent) for 1 hour(s) and 10 minute(s) · Concentration: 2% · Temperature: 37°C

Giovanna Roncador

Verified customer

Submitted Jul 24 2015

Application
Immunocytochemistry/ Immunofluorescence
Blocking step
BSA as blocking agent for 12 hour(s) and 0 minute(s) · Concentration: 3% · Temperature: 4°C
Sample
Mouse Cell (oocyte)
Specification
oocyte
Permeabilization
Yes - 0.01% triton-X100
Fixative
Paraformaldehyde

Abcam user community

Verified customer

Submitted Apr 28 2014

Answer

Merci de nous avoir contactés.

En compensation des 4 unités de ab10579 qui n'ont pas fonctionné, j'ai mis en place l'envoi d'une unité de ab32084 et d'une unité de ab1838, les numéros de commandes sont ******* et ***** (consolidées pour former un seul colis). J'ai également demander à notre service financier d'éditer un avoir pour 2 unités de ab10579. La référence de cet avoir est CN ********.

Voici les manières dont vous pouvez utiliser cette référence CN:
(1) la faire valoir contre la facture originale si celle-ci n'as pas encore été payée,
(2) la faire valoir contre une prochaine facture,
(3) obtenir un remboursement de la somme entière si vous n'avez pas de factures actuellement en cours avec Abcam,

Si vous souhaitez recevoir un remboursement au lieu d'un avoir, veuillez demander à votre service de gestion de prendre contact avec notre département comptabilité afin que nous puissions recueillir les informations nécessaires pour cette restitution. Notre service comptabilité peut être contacté par courriel à l'adresse mailto:creditcontrol@abcam.com ou par téléphone en utilisant le lien « Contactez-nous » dans le coin en haut à droite de notre site internet. Veuillez communiquer votre numéro d'avoir lorsque vous contactez notre service comptabilité.

Vous recevrez également la référence complète de la note de crédit par courriel ou par voie postale; cette référence commençant par les lettres CGB.

N'hésitez pas à nous contacter de nouveau si vous avez d'autres questions.

Read More

Answer

Thank you for your reply.

I hope the new vial will provide good results!

I am sorry that I have not seen the crossreactivity note on the datasheet of the secondary antibody.I might would nevertheless performa control slide using your settings/antibodies.

In regards to the PFA fixation, it might well depend on the cell and other antibodies, whether this is suitable. I do however not think that the storage is incompatible with PFA - you fixed the cells for about 10 minutes, washed them, and keep them in PBS/Azide in the fridge? This normally should work. In contrast to Methanol fixation, PFA fixation requires cell permeabilization after fixation. Maybe thepermeabilization step canbe improved,up to 0.2% Triton in PBS can be used for 10 minutes.

As said above, I hope the new vial will resolve the problem. Please keep us updated and do not hesitate to contact us also meanwhile, should you have any questions or concerns. I look forward hearing back from you!

Read More

Question

3) Staining is not strictly nuclear, without appearance of foci as it should

4) Sample preparation:
Human glioblastome cell line (U87MG)
Cells were fixed 20 minutes with cold methanol and store 4 weeks at -20°C. (Samples can be stored 8 weeks until deterioration)

5) Fixation step
Yes
If yes: methanol 100%
20 minutes at room temperature

6) Antigen retrieval method (if applicable) NO

7) Permeabilization method:
The permeabilization is realized during blockage with the same reagent. Permeabilization with Triton X-100 0.5% diluted in PBS 0.8X, NaCl 50mM and 3% of milk. The permeabilization/blacage is carried out for one hour at room temperature

9) Endogenous peroxidases blocked? NO
Endogenous biotins blocked? NO
10) Primary antibody (If more than one was used, describe in “additional notes”) :
Dilution : 1/100 on blocking buffer

The staining buffer is the same that blocking reagent. The staining is carried out overnight at 4°C in wet room

11) Secondary antibody:
Donkey anti mouse Al 568 (Invitrogen) was used at dilution of 1/1000 in the blocking buffer during one hour at room temperature


12) Washing after primary and secondary antibodies:
After the fist antibody, cells were washed three times during 10min (each) with PBS0.8X, NaCl 50mM and Milk 1.5%.
After the second antibody, cells were washed three times during 10 minutes (each) with PBS 0.8X, NaCl 50mM, Tritton X-100 at 0.1%.


13) the staining is observed with fluorescence microscopy excited at 579nm


14) I have made this protocol many times (with your antibody) and it was the first time that staining look like aspecific
Usually, I’ve always seen nuclear spots with poor background noise
This protocol was still validated and published before I use it. I don’t optimized it

Additional notes:
I always used your TRF-1 antibody in co-staining with Rabbit 53BP1 antibody (NOVUS). The 53BP1 antibody is used at same dilution, in same buffer and in same time than TRF1 antibody. To labeled the 53BP1 antibody, Donkey anti-rabbit Al488 was used, observed with fluorescence microscopy excited at 499nm

Read More
Answer

Thank you very much for having provided all these additional information.

I have seen the results and I agree that the staining is not as the previous staining. As for the reasons explained before, I just would like to ask you to confirm the two following points:

1.) Can you confirm that the secondary antibody used is not crossreacting with the primary rabbit antibody? Indeed, I have looked at the datasheet of the secondary antibody, and I could not find the confirmation that this antibody had been crossabsorbed. Therefore I strongly recommend to check whether the secondary anti mouse antibody does not recognize also the rabbit primary antibody.

2.) Most of the stainingI have seen for TRF1 have been made with PFA. I agree that the protocol you used has been well established. However as a general note, the Methanol does precipitate proteins and can therefore result in a staining with lower resolution. If the resolution is important for your analysis, it might be worth to check a PFA staining.

I have arranged now the shipping of a vial of a new batch of the ab10579 to the address you provided. You will shortly receive also a confirmation email with the shipping details.

Please let me know if you have any questions or concerns. I would also appreciate if you could provide us with the feedback whether the new vial has provided satisfactory results. I look forward hearing back from you!

Read More

Answer

Thank you for contacting us.

While to our knowledgethis TRF1 antibody has not been tested for cross-reactivity with TRF2, it is not likely to detect that protein. The homology between TRF1 and TRF2 is only around 17%. We have not had any customers report cross-reactivity between these proteins.

Based on the information available in the UniProt database (linked below), TRF1 and TRF2 can sometimes be found in the same protein complex. If you are detecting a complex, it is possible that you might get the same band with both antibodies, even if they are specific for the correct proteins. To ensure that this isn't the case, you may want to make sure you are running a reduced and denatured blot and boiling for a sufficient amount of time. I typically recommend boiling for 5-10 minutes in the presence of BME or DTT, but if you are concerned about complexes, you can increase this to 15 minutes.

http://www.uniprot.org/uniprot/P54274

I hope this will help to improve your results, if not, please let me know what protocol you have been using and I will be happy to help you further.

Read More

1-10 of 21 Abreviews or Q&A

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