Overview

  • Product name
    Anti-TRF1 antibody - ChIP Grade
    See all TRF1 primary antibodies
  • Description
    Rabbit polyclonal to TRF1 - ChIP Grade
  • Host species
    Rabbit
  • Tested applications
    Suitable for: ChIP, WB, ICC/IF, IPmore details
    Unsuitable for: IHC-P
  • Species reactivity
    Reacts with: Mouse, Human
  • Immunogen

    Human full length recombinant protein expressed in bacteria.

  • Positive control
    • HL60 WCE lysate

Properties

Applications

Our Abpromise guarantee covers the use of ab1423 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
ChIP Use at an assay dependent dilution. PubMed: 1946086
WB 1/2000. Detects a band of approximately 60-65 kDa. An additional non specific band at 35 kDa may be observed. Denatured whole cell extracts (prepare total cell extracts with 6M urea, 1% SDS, 150 mM NaCl, 25mM Tris ph8, and then sonicate for 10 seconds)and TBS based buffers with 0.5% Tween 20 are recommended.
ICC/IF Use at an assay dependent dilution. PubMed: 20427319
IP Use at an assay dependent dilution.
  • Application notes
    Is unsuitable for IHC-P.
  • 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

    • Western blot using anti-TRF1 at 1/2000. A single band at 60-65 kD is detected. Western blot using anti-TRF1 at 1/2000. A single band at 60-65 kD is detected.
    • Cells transfected with a TRF1-GFP contruct. On the left: immunofluorescence with the anti-TRF1 antibody used at 1/300, with a Texas Red-conjugated secondary antibody. In the centre: fluorescence from the GFP. Right: merge of both images showing overlap of GFP fluorescence and antibody staining.

    • The cells in the first image are probably in a stage of the cell cycle where the signal is not so clear. In this image the antibody is shown to recognise the same signal but staining is shown as 20-30 spots (punctate foci).

    References

    This product has been referenced in:
    • Margalef P  et al. Stabilization of Reversed Replication Forks by Telomerase Drives Telomere Catastrophe. Cell 172:439-453.e14 (2018). Mouse . Read more (PubMed: 29290468) »
    • Ling X  et al. TERT regulates telomere-related senescence and apoptosis through DNA damage response in male germ cells exposed to BPDE in vitro and to B[a]P in vivo. Environ Pollut 235:836-849 (2018). Mouse . Read more (PubMed: 29353801) »
    See all 30 Publications for this product

    Customer reviews and Q&As

    1-10 of 16 Abreviews or Q&A

    Answer

    Thank you for your reply.

    Our datasheet for this antibody does not suggest any specific ICC protocol, as a general ICC protocol should work with this antibody. The protocol stated on the datasheet belongs to a customer’s review who submitted an Abreview for this antibody in that application, and that can actually serve as guidance for other researches.

    I understand, however, that this antibody should have given the expected results under the conditions you have used it, and it may well be that you have received a faulty vial.

    If the antibody is still under the 6 months guarantee I will be happy to either send you a replacement for the same or a different antibody, or, as you mentioned in your last email, refund the product’s price.

    Could you please send me your order number to be able to raise the replacement / refund?

    Thank you very much for your cooperation. I look forward to receiving your reply with further details.

    Read More
    Application
    Western blot
    Loading amount
    50 µg
    Gel Running Conditions
    Reduced Denaturing (8% resolution gel)
    Sample
    Human Cell lysate - whole cell (hepatocellular carcinoma)
    Specification
    hepatocellular carcinoma
    Treatment
    Inhibition of one gene's expression using lentiviral shRNA
    Blocking step
    Milk as blocking agent for 3 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 24°C

    Abcam user community

    Verified customer

    Submitted Jul 16 2013

    Question
    Answer

    Thank you for contacting us and bringing this to our attention. The switch to PFA fixation and detergent permeabilization instead of methanol fixation that I mentioned in our conversation may help, as that is what has worked for customers in the past.

    Please let us know if you continue to have difficulty.

    Read More

    Answer

    Thank you for confirming these details and for your cooperation. The details provided enable us to closely monitor the quality of our products.

    I am sorry this product did not perform as stated on the datasheet and for the inconvenience this has caused. As requested, I have issued a free of charge replacement with the order number 1016117.

    To check the status of the order please contact our Customer Service team and reference this number.

    Please note that this free of charge replacement vial is also covered by our Abpromise guarantee. Should you still be experiencing difficulties, or if you have any further questions, please do not hesitate to let us know.

    I wish you the best of luck with your research.

    Read More
    Abcam guarantees this product to work in the species/application used in this Abreview.
    Application
    Immunocytochemistry/ Immunofluorescence
    Sample
    Human Cell (Breast carcinoma)
    Specification
    Breast carcinoma
    Fixative
    Paraformaldehyde
    Permeabilization
    Yes - 0.5% Triton X-100
    Blocking step
    Serum as blocking agent for 30 minute(s) · Concentration: 2% · Temperature: 37°C

    Abcam user community

    Verified customer

    Submitted Aug 23 2009

    Application
    Western blot
    Sample
    Human Cell lysate - whole cell (HeLa cell)
    Loading amount
    100 µg
    Specification
    HeLa cell
    Gel Running Conditions
    Reduced Denaturing
    Blocking step
    Milk as blocking agent for 30 minute(s) · Concentration: 5%

    Dr. Geun Hyoung Ha

    Verified customer

    Submitted May 27 2009

    Application
    Western blot
    Sample
    Human Cell lysate - whole cell (Hela CELLS)
    Loading amount
    50 µg
    Specification
    Hela CELLS
    Gel Running Conditions
    Reduced Denaturing (8%)
    Blocking step
    Milk as blocking agent for 30 minute(s) · Concentration: 5% · Temperature: 25°C

    Mr. Geun-Hyoung Ha

    Verified customer

    Submitted Apr 30 2009

    Question

    follow up to previous correspondance: 1) Did the customer aliquot the vial upon receipt and uses a fresh aliquot every time? -> yes!! 2) Is the HeLa cell extract a nuclear extract? The protein is a nuclear protein and absence in the WB could be due to the low amount of TRF1 in the whole cell lysate. -> whole cell extract. but I tested TRF2 of other company, I detected TRF2 very well with whole cell extract. 3) Were the protease inhibitors added freshly to the lysis buffer? Were samples kept on ice at all times? -> of course! PIC was added and all samples kept on ice well. 4) Was the sample mixed with loading buffer and boiled? -> yes 5) You say "amount protein loaded: 50ug" is this for the WB only or for the IP too? -> No, I used 2mg for IP. Input was 50ug of protein. 6) Has the customer tried 5% BSA block? -> yes, but same result. 7) has the customer tried a higher dilution with the IPed samples? -> of course. I tried 1/500, 1/1000, 1/2000, 1/5000. 8)What is the buffer used to dilute the primary antibody and the secondary antibody? -> 5% milk(5% BSA also) and TBST only. 9) The WB image you provide says "TRF1 is not detected but orc4 is detected well". I see a faint band around the 55kDa mark which could be TRF1. The Swiss Prot database indicates that the MW of TRF1 is around 50kDa. The customer is happy with the faint band of orc4 but I would disagree and optimise the protocol for both antibodies. -> orc4 band was not faint. just scanner was bad.. and other our lab members see orc4 everyday. I think TRF1 antibody (delivered to me) has something wrong. 10) When investigating low molecular weight proteins the customer should use high percentage gels, use of a 7% gel means the bands are at the bottom of the gel and not well separated. -> but I detected well orc4(40kDa) and other proteins of low molecular (lower than TRF1) with 7% SDS gel. also, I tried again with 12% gel. but result was bad.. 11) for investigating the problem with the IP, I would need a detailed protocol of the IP. I suggest we first concentrate on the WB to find out if the antibody works. I believe the faint 50kDa band might be the TRF1 but an optimised protocol is necessary to get a clean band. The background from the IP samples could be due to the blocking agent used, the type of protein A or G beads used for the IP and other factors. Has the customer much experience of IP? -> 1 tube : whole cell extracts(1mg, 2mg of protein) were incubate with protein A beads (G beads also) for pre-clearing. 2 tube : lysis buffer and TRF1 antibody (diluted 1/500,1/1000, 1/2000, 1/5000.) were incubate with protein A beads (G beads also) for TRF1 antibody can attach to beads. ... after that, pre-cleared cell extract of 1 tube was transfered to protein A beads attached TRF1 antibody of 2 tube and incubated for 2,3,4,5hrs and O/N.

    Read More
    Answer

    Thank you for this information, it is very useful and I can now really understand the protocol and the optimisation steps. I think the WB protocol looks very good (apart from my suggestion of running a high percentage gel), the IP protocol we suggest is very different but the fact the customer has a problem in WB means the problem is not related to the IP protocol. I understand that the customer can detect TRF2 in whole cell extracts, but it is possible that the TRF1 levels are much lower and a nuclear extraction would concentrate the protein A/G-sepharose beads for 4 hours and finally boiling the sample . If you can provide me with the purchase order or Abcam order number I can send you a replacement antibody to try, but I would strongly recommend nuclear extraction to maximise the amount of TRF1 protein loaded on the gel. I would also suggest changes to the IP protocol: incubate the cell extracts with the antibody overnight (try 5ug antibody per 500ug lysate), then the next morning add the protein A sepharose beads, incubate together for 4hrs, then wash the beads and boil, separating the protein from the antibody and from the beads. Please do not hesitate to contact us for further details of our recommended IP protocol. I look forward to hearing from you,

    Read More

    Answer

    I have carefully looked at the protocol and unfortunately need more information to understand the problem, I am sorry for the delay. The antibody has not been tested on HeLa cells, it does cross react with human but it is known that cancer cells can be very different morphologically and biochemically, this may explain why the signal is low. 1) Did the customer aliquot the vial upon receipt and uses a fresh aliquot every time? 2) Is the HeLa cell extract a nuclear extract? The protein is a nuclear protein and absence in the WB could be due to the low amount of TRF1 in the whole cell lysate. 3) Were the protease inhibitors added freshly to the lysis buffer? Were samples kept on ice at all times? 4) Was the sample mixed with loading buffer and boiled? 5) You say "amount protein loaded: 50ug" is this for the WB only or for the IP too? 6) Has the customer tried 5% BSA block? 7) has the customer tried a higher dilution with the IPed samples? 8)What is the buffer used to dilute the primary antibody and the secondary antibody? 9) The WB image you provide says "TRF1 is not detected but orc4 is detected well". I see a faint band around the 55kDa mark which could be TRF1. The Swiss Prot database indicates that the MW of TRF1 is around 50kDa. The customer is happy with the faint band of orc4 but I would disagree and optimise the protocol for both antibodies. 10) When investigating low molecular weight proteins the customer should use high percentage gels, use of a 7% gel means the bands are at the bottom of the gel and not well separated. 11) for investigating the problem with the IP, I would need a detailed protocol of the IP. I suggest we first concentrate on the WB to find out if the antibody works. I believe the faint 50kDa band might be the TRF1 but an optimised protocol is necessary to get a clean band. The background from the IP samples could be due to the blocking agent used, the type of protein A or G beads used for the IP and other factors. Has the customer much experience of IP? I look forward to hearing from you, Thank you for your patience,

    Read More

    1-10 of 16 Abreviews or Q&A

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