Anti trf2

Manufactured by Merck Group

Sourced in United States

Anti-TRF2 is a laboratory reagent used to detect and quantify the expression of the TRF2 protein, which is involved in the protection and maintenance of telomeres. It is a specific antibody that can be used in various analytical techniques, such as Western blotting, immunoprecipitation, and immunohistochemistry, to study the role of TRF2 in cellular processes.

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7 protocols using anti trf2

ChIP-seq Antibodies and Immunoblotting Protocols

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For ChIP, normal rabbit IgG (Santa Cruz, sc-2027), anti-histone H3 (Abcam, ab1791), anti-TRF1 (in-house), anti-TRF2 (Millipore, #05-521), anti-TPP1 (in-house) and anti-Pol II C-terminus domain (CTD) (Abcam, ab5408; clone 4H8) were used. Anti-phospho Ser-2 CTD (clone 3E10) and anti-phospho Ser-5 CTD were previously described [27] (link). Immunoblotting utilized anti-nucleolin (Abcam, ab13541), anti-tubulin (Sigma, T3526), anti-hnRNPA1 (Abcam, ab10685) anti-histone H2B (Millipore, #07-371), and anti-GAPDH (Millipore, clone 6C5).

Wakai M., Abe S., Kazuki Y., Oshimura M, & Ishikawa F. (2014). A Human Artificial Chromosome Recapitulates the Metabolism of Native Telomeres in Mammalian Cells. PLoS ONE, 9(2), e88530.

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Western Blot Analysis of DNA Damage Response

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Western blot analysis was performed as previously reported (36 (link)). Expression levels of TRF2 were evaluated by using the mouse mAb anti-TRF2 (Millipore). The DNA damage response was evaluated by using the following antibodies: rabbit mAb anti-Ser1981 p-ATM (Abcam Ltd.); rabbit pAb anti-Thr68 p-CHK2 (Cell Signaling, Beverly, MA, USA); mouse monoclonal anti-γH2AX antibody (Upstate, Lake Placid, NY, USA).
The purity of the subcellular fractions was evaluated by using the following antibodies: mouse mAb anti-EGFR (a kind gift of Dr Oreste Segatto, Regina Elena National Cancer Institute of Rome); mouse mAb anti-Lamin A/C (636, Santa Cruz Biotecnology); mouse mAb anti-GAPDH (clone 6C5, Santa Cruz Biotecnology).

Zizza P., Dinami R., Porru M., Cingolani C., Salvati E., Rizzo A., D’Angelo C., Petti E., Amoreo C.A., Mottolese M., Sperduti I., Chambery A., Russo R., Ostano P., Chiorino G., Blandino G., Sacconi A., Cherfils-Vicini J., Leonetti C., Gilson E, & Biroccio A. (2019). TRF2 positively regulates SULF2 expression increasing VEGF-A release and activity in tumor microenvironment. Nucleic Acids Research, 47(7), 3365-3382.

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Immunofluorescence Staining Panel

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Anti-lamin A/C (Santa Cruz, sc6215, 1:1000 and Cell Signaling Technology, 2032T, 1:1000); anti-BrdU (Becton Dickinson, 347580, 1:20), anti-Ki67 (Abcam, ab16667, 1:50); anti-TRF2 (Millipore, 05–521, 1:200); anti-Tubulin (Sigma-Aldrich, T5168, 1:2000); anti-HP1α (Sigma-Aldrich, H2164, 1:2000); anti-H3K9me3 (Millipore, 05–1242, 1:2000); anti-lamin B1 (Abcam, ab16048, 1:5000); anti-p16 (Santa Cruz Biotechnology, sc-1207, 1:800); anti-Keratin5 (BioSite, PRB-160P, 1:500 and Abcam, ab52635, 1:100); anti-phospho KAP-1 (S824) (Bethyl Laboratories, A300–767A, 1:200); anti-53BP1 (Novus Biologicals, NB100–304, 1:1000), Anti-CD45, (Abcam, ab10558, 1:500).

Aguado J., Sola-Carvajal A., Cancila V., Revêchon G., Ong P.F., Jones-Weinert C.W., Wallén Arzt E., Lattanzi G., Dreesen O., Tripodo C., Rossiello F., Eriksson M, & d’Adda di Fagagna F. (2019). Inhibition of DNA damage response at telomeres improves the detrimental phenotypes of Hutchinson–Gilford Progeria Syndrome. Nature Communications, 10, 4990.

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Characterization of HP1BP3 Protein

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All cells were cultured in DMEM supplemented with 10% fetal bovine serum and 100 units/ml penicillin/streptomycin at 37°C and 5% CO₂. Human full-length HP1BP3 cDNA was cloned into pDEST27 (Invitrogen) for GST tagging and pHAGE-based vectors for Flag tagging (Addgene). pET-MBP-His6-based vectors were for MBP tagging (Addgene). HP1 α cDNA was cloned into pHAGE-based vectors for Flag tagging (Addgene). Histone H1C recombinant protein was purchased from company (Sigma, H1917). Cells were reverse transfected with siRNAs for 48–72 h before analysis. The siRNA sequences are:
Antibodies used in the study include: rabbit polyclonal anti-HP1BP3 (generated in the lab), rabbit polyclonal anti-Flag (Sigma, F7425), anti-HA (Sigma, H3663), anti-GAPDH (ABclonal, AC027), anti-TRF2 (Millipore, 05-521), anti-γH2AX (Millipore, 05-636), rabbit polyclonal anti-GST (Abmart, M20007), mouse monoclonal anti-PML (Santa Cruz, sc966), rabbit polyclonal anti-Histone H3 (Abcam, ab1791), rabbit polyclonal anti-H3K9me3 (Abcam, ab8898) and rabbit polyclonal IgG (Millipore, 12-370). Monoclonal anti-TIN2 antibodies were generated in the Songyang lab.

Shi G., Hu Y., Zhu X., Jiang Y., Pang J., Wang C., Huang W., Zhao Y., Ma W., Liu D., Huang J, & Songyang Z. (2020). A critical role of telomere chromatin compaction in ALT tumor cell growth. Nucleic Acids Research, 48(11), 6019-6031.

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Telomeric DNA Analysis by ChIP-blot

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Cells were cross-linked with 1% formaldehyde for 10 min at room temperature and washed twice with cold PBS, resuspended in SDS lysis buffer (50mM Tris–HCl, pH = 8.1, 10 mM ethylenediaminetetraacetic acid, 1% SDS) and sonicated to fragments of 200 bp to 1 kb. The supernatant was pre-cleared with Protein-A agarose beads precoated with Escherichia coli genomic DNA. Chromatin immunoprecipitation (ChIP) was carried out overnight at 4°C with anti-TRF2 (Millipore), anti-H3K9ac (Sigma), anti-H4ac (Millipore) or IgG (Sangon). Beads were washed three times, and eluted with 0.1M NaHCO₃ & 1% SDS, followed by reverse cross-linking and phenol-chloroform extraction. DNA fragments were precipitated by ethanol and blotted onto NC membranes. Telomeric DNA was detected by hybridization with telomere-specific probe.

Ge Y., Wu S., Xue Y., Tao J., Li F., Chen Y., Liu H., Ma W., Huang J, & Zhao Y. (2016). Preferential extension of short telomeres induced by low extracellular pH. Nucleic Acids Research, 44(17), 8086-8096.

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Western Blotting Analysis of TRF2 and DNA Damage

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Total cell lysates were prepared using a proper buffer (50 mmol/l Tris–HCl pH 7.5, 5 mmol/l EDTA, 250 mmol/l NaCl, 0.1% Triton) containing protease and phosphatase inhibitors (Thermo Fisher Scientific). Samples were sonicated (Diagenode, UCD‐200‐TMEX‐Bioruptor Standard) followed by centrifugation at 16,000 g for 2 min. Supernatants were recovered and used for Western blotting analysis. Expression levels of TRF2 were evaluated by using anti‐TRF2 (1:1,000, Millipore, 4A794), anti‐TRF1 (N‐19) (1:1,000, Santa Cruz Biotechnology, Santa Cruz, CA, USA). The DNA damage response was evaluated by using the following antibodies: p‐ATM (Ser1981) (1:1,000, Cell Signaling Technology, Beverly, MA, USA), anti‐ATM (1:1,000, Cell Signaling Technology, Beverly, MA, USA); anti‐γH2AX (Ser139) (1:2,000, Millipore, Bedford, MA). Actin signal was detected by mouse anti‐β‐actin (1:10,000, Sigma Aldrich), and it was used as loading control. The protein band intensity was quantified by densitometry analysis using ImageJ software.

Dinami R., Pompili L., Petti E., Porru M., D'Angelo C., Di Vito S., Rizzo A., Campani V., De Rosa G., Bruna A., Serra V., Mano M., Giacca M., Leonetti C., Ciliberto G., Tarsounas M., Stoppacciaro A., Schoeftner S, & Biroccio A. (2022). MiR‐182‐3p targets TRF2 and impairs tumor growth of triple‐negative breast cancer. EMBO Molecular Medicine, 15(1), e16033.

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Immunofluorescence Assay for SKIV2L, TTC37, WDR61, and TRF2

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Cells were grown on 4-well culture slides, pre-extracted for 5 min with permeabilisation buffer (50 mM NaCl, 3 mM MgCl2, 20 mM Tris pH 8, 0.5% (v/v) Triton X-100, 300 mM sucrose), fixed for 15 min in fixative solution (formaldehyde 4% (w/v) / sucrose 2% (w/v)) and permeabilised for another 10 min. Slides were incubated for 30 min with the blocking buffer (10% (v/v) goat or donkey serum (Stratech Scientific Ltd) in 1X PBS) at 37°C. Then, the primary antibody (anti-SKIV2L, Proteintech group, 11462-1-AP, 1:1000; anti-TTC37, Novus Biologicals, NBP1-93640, 1:500; anti-WDR61, Sigma-Aldrich, SAB1401852, 1:750 or anti-TRF2, Millipore, 05-521, 1:500) was added in blocking buffer and incubated for 1 hour at 37°C. After 3 x 3 min washes with 1X PBS, the secondary antibody (1/400 in blocking buffer, goat anti-rabbit Alexa 594 antibody, Thermo Fisher, A-11037 or donkey anti-mouse Alexa 488 antibody, Thermo Fisher, R37114) was added in blocking buffer for 30 min at 37°C, followed by 3 x 3 min washes with PBS 1X. Finally, mounting media containing DAPI (Prolong gold antifade mountant with DAPI, Invitrogen, P36931) was used to counterstain DNA.

Herrera‐Moyano E., Porreca R.M., Ranjha L., Skourti E., Gonzalez-Franco R., Sun Y., Stylianakis E., Montoya A., Kramer H., & Vannier J. (2020). The human SKI complex prevents DNA-RNA hybrid-associated telomere instability.

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