GFP (Roche, 11814460001; 1:2000), PAR 10H (Trevigen, 4335-AMC-050; 1:200), PAR binder (Millipore, MABE1031, 1:1000), CTIF (Sigma-Aldrich, HPA016865-100UL; 1:1000), TERF1 (Abcam, ab10579; 1:1000), GLUT4 (Abcam, ab654; 1:1000), ILF3 (Abcam, ab92355; 1:1000), NPM1 (Abcam, ab10530; 1:1000), CETN3 (Abnova, H00001070-M01; 1:500), PCNT (Atlas antibodies, HPA016820; 1:500), PCM1 (Cambridge bioscience, A301-149A; 1:500), gamma-tubulin (Sigma-Aldrich, T6557-100UL; 1:500), Azi1/Cep131 (Abcam, ab84864; 1:500), Cep290 (Abcam, ab84870; 1:500), BBS4 (Proteintech, 12766-1-AP; 1:500), OFD1 (Proteintech, 22851-1-AP; 1:500), TNKS1/2 (Santa Cruz, sc-8337; 1:1000), alpha-tubulin (Sigma-Aldrich, T9026; 1:10000), beta-actin (Sigma-Aldrich, A1978; 1:10000).
Ab10579
Manufactured by Abcam
Sourced in United Kingdom
Ab10579 is a laboratory equipment product offered by Abcam. It is a device designed for a specific core function within the scientific research process. No further details can be provided while maintaining an unbiased and factual approach.
Automatically generated - may contain errors
Lab products found in correlation
Bovine serum albumin (bsa), by Merck Group (2 mentions)
A1978, by Merck Group (1 mentions)
Alpha tubulin, by Merck Group (1 mentions)
Gamma tubulin, by Merck Group (1 mentions)
Ab654, by Abcam (1 mentions)
Ab92355, by Abcam (1 mentions)
β actin, by Merck Group (1 mentions)
Img 124, by Novus Biologicals (1 mentions)
Dapi fluoromount g, by Southern Biotech (1 mentions)
Dm2500 microscope, by Leica (1 mentions)
Duolink 2 fluorescence, by Olink (1 mentions)
Cd90 5e10, by BD (1 mentions)
Cd38 hit2, by BD (1 mentions)
Nb100 304, by Abcam (1 mentions)
Prolong gold antifade reagent, by Thermo Fisher Scientific (1 mentions)
A21207, by Thermo Fisher Scientific (1 mentions)
A11029, by Thermo Fisher Scientific (1 mentions)
Plan apo 60x, by Nikon (1 mentions)
A1rsi confocal microscope, by Nikon (1 mentions)
Vectashield mounting medium, by Vector Laboratories (1 mentions)
13 protocols using ab10579
1
Immunofluorescence Protein Detection Protocol
2
Protein-Protein Interaction Detection Using Duolink PLA
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PLA (Duolink II Fluorescence, Olink Bioscience) was used for the detection of protein-protein interactions according to the manufacturer's instructions. Briefly, adherent cells grown on round glass slides in 24-well plates were fixed for 10 min with 4% paraformaldehyde, permeabilized in PBS containing 0.5% Triton X-100 for 20 min at room temperature, and then blocked by incubation in PBS containing 7.5% goat serum and 7.5% foetal bovine serum for 1 h at room temperature. Next, the cells were incubated overnight at 4°C with two antibodies against the two proteins of interest. The following primary antibodies were used: TRF2 (1:100, IMG-124, Imgenex), PCAF (1:100, AB9962, Millipore), GCN5 (1:100, H-75, Santa Cruz), TRF1 (1:100, ab10579, Abcam), and P300 (1:100, NA 46, Calbiochem). The slides were then washed three times with 0.1% Triton X-100 in PBS and incubated with two PLA probes (PLA probe MINUS stock and PLA probe PLUS stock; Duolink II) for 1 h at room temperature. Subsequently, the slides were washed three times with 0.1% Triton X-100 in PBS. Ligation was conducted for 30 min at 37°C, followed by two washes with 0.1% PBS and Triton X-100 and amplification using DNA polymerase (Duolink II) for 100 min at 37°C. The slides were then washed, dried and mounted with Dapi-Fluoromount-G (Southern Biotech). Images were captured using a Leica DM 2500 microscope.
3
Multiparameter Flow Cytometry and Cell Sorting
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The following monoclonal Abs were used for flow cytometry and cell sorting: anti-CD34 (581, BD Biosciences, 1:5), -CD38 (HIT2, BD Biosciences, 1:5), -CD45RA (HI100, BD Biosciences, 1:20), -CD90 (5E10, BD Biosciences, 1:20), -CD49f (GoH3, Biolegend, 1:5), and -CD45 (HI30, BD Biosciences, 1:20). A mixture of biotin-conjugated anti-CD2, -CD3, -CD11b, -CD14, -CD15, -CD16, -CD19, -CD56, -CD123, and -CD235a (130-092-211, Lineage Cell Depletion Kit human, Miltenyi Biotec Inc., 1:5) was used as the lineage mix. The following Abs were used for immunocytochemistry: anti-human TRF1 (TRF-78, ab10579, Abcam, 1:100), 53BP1 (NB100-304, 1:200), and RPA32 (sc-28709, 1:200).
4
Quantifying DNA Damage and Telomere Dysfunction
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DNA damage and telomere dysfunction were quantified as in previous studies via the detection of γH2AX foci corresponding to phosphorylated histone H2AX and telomere dysfunction-induced foci (TIFs) corresponding to regions wherein γH2AX foci co-localize with telomere repeat binding factor-1 (TRF-1) [35 (link)]. Briefly, cells that had been plated onto coverslips were first permeabilized for 10 minutes using 0.25% Triton X-100, after which they were fixed for 15 minutes using 4% PFA, re-permeabilized with Triton X-100, and blocked using 1% BSA (Sigma). Samples were then probed for 1 h with anti-TRF-1 (#ab10579, 1:500, Abcam) and anti-γH2AX (#9718, 1:200, Cell Signaling), followed by incubation with appropriate AF488- and AF594-conjugated IgGs, respectively (#A-11029, 1:500; # A-21207, 1:1000; Invitrogen). Samples were then washed with PBST, nuclei were counterstained using DAPI, and the ProLong Gold Antifade reagent (Invitrogen) was used to mount samples prior to their analysis via immunofluorescence confocal microscopy (Nikon Inc., Tokyo, Japan). ImageJ was used to quantify the average number of γH2AX foci, TRF-1 foci and TIFs (γH2AX foci co-localized with TRF-1 foci) per cell.
5
Immunofluorescence Staining of NCAPH2 and TRF1
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Cells grown on coverslips in 12‐well plates were fixed 100% methanol on ice for 3 min, permeabilized in 0.5% Triton X‐100 in PBS, and washed three times in PBS + 0.1% Triton X‐100 (PBST). Cells were blocked in 5% bovine serum albumin (BSA) in PBST and incubated with the following primary antibodies in PBST + 5% BSA overnight at 4°C: rabbit anti‐NCAPH2 (1:200) and mouse anti‐TRF1 (ab10579, 1:200, Abcam). Coverslips were washed three times in PBST and incubated with secondary antibodies conjugated with Cy3 or Alexa488 (1:500, Jackson Laboratories, West Grove, PA). Nuclei were counterstained with 0.1 μg/μl 4′, 6‐diamidino‐2‐phenylindole (DAPI) and coverslips were mounted in Vectashield Mounting medium (Vector Laboratories, Burlingame, CA). Slides were imaged using a Nikon A1RSi confocal microscope with Plan Apo 60X and 100X oil immersion objectives and the Nikon Elements 4.0 software package (Nikon, Melville, NY). Fluorescence intensity was measured on a single z‐stack slice using ImageJ and plots were generated in Microsoft Excel.
6
Immunofluorescence Staining of Cellular Proteins
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Slides were either fixed with MeOH at −20 °C or with 4% formaldehyde at room temperature (RT) for 10 to 15 min, and were then incubated with blocking buffer (0.8 × PBS, 50 mM NaCl, 0.5% Triton X 100, 3% milk) for 1 h, followed overnight by incubation at 4 °C with primary antibody to TRF2 (ab217529, 1/100, abcam), to p65 (#8242, 1/200, CST), to γ-H2AX (ab2893, 1/100, abcam) and mouse polyclonal antibody to TRF1 (ab10579, 1/100, abcam) in 0.8 × PBS, 50 mM NaCl, 0.5% Triton X 100, and 3% milk. Cells were then washed three times for 10 min in 0.8 × PBS, 50 mM NaCl, and 1.5% skimmed milk at RT. Incubation with donkey polyclonal anti-mouse ALEXA488 (A21202; Molecular Probes) and donkey polyclonal anti-rabbit ALEXA555 (A31752; Molecular Probes) antibodies was performed for 1 h at 37 °C in the dark in 0.8 × PBS, 50 mM NaCl, 0.5% Triton X 100, and 3% skimmed milk. All antibody incubations were performed in a moist chamber. Cells were then washed three times for 10 min in 0.8 × PBS, 50 mM NaCl, and 0.5% Triton X 100. Slides were then rinsed in PBS, counterstained with 4′,6-diamidino-2-phenylindole (DAPI), mounted in VECTASHIELD, and stored at 4 °C in the dark.
7
Quantifying DNA Damage Markers by Microscopy
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The foci assay for γH2AX, 53BP1, and Trf1 was performed as previously described [26 (link)]. Briefly, cells grown on coverslips were fixed, blocked, and stained for γH2AX and 53BP1 or γH2AX and Trf1. Foci for γH2AX and 53BP1 were assessed by LSM using a single focus plane; for γH2AX and Trf1 quantification, the whole cell was screened, and images were stacked. For each experiment, at least 100 cells were analyzed. Primary antibodies used were γH2AX (1:500, rabbit, Cell Signaling Technology, MA, USA, mAb #9718S), γH2AX (1:500, mouse, Merck Millipore, Darmstadt, Germany, 05-636), 53BP1 (1:500, mouse, Sigma Aldrich, Karlsruhe, Germany, MAB 3802), and Trf1 (1:500, mouse, Abcam, Cambridge, UK, ab10579). Secondary antibodies used were Alexa Fluor 488 goat anti-mouse (1:1000, Thermo Fisher Scientific Invitrogen, Waltham, USA, A11017) and Cy3 goat anti-rabbit (1:1000, Abcam, ab97075). The software ImageJ (Wayne Rasband, NIH, Bethesda, MD, USA) was used to count foci and measure colocalization.
8
In Situ Proximity Ligation Assay for NCAPH2, TRF1, and PML
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In situ proximity ligation assays (PLAs) were performed using the Duolink In Situ Red Starter Kit (Sigma‐Aldrich, Natick, MA). RPE‐1 cells were fixed in 4% formaldehyde in 1X PBS for 10 min, permeabilized in 0.5% Triton X‐100 in PBS, and washed three times in PBS + 0.1% Triton X‐100 (PBST). Cells were blocked in 5% BSA in PBST and incubated with the following primary antibodies in PBST + 5% BSA overnight at 4°c: rabbit anti‐NCAPH2 (1:200), mouse anti‐TRF1 (ab10579, 1:200, Abcam), and rabbit anti‐PML (sc‐5621, 1:100, Santa Cruz). PLA probe incubation, ligation, amplification, and detection were carried out according to the manufacturer's instructions and slides were imaged as above.
9
Antibody Sources and Characteristics
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Rabbit antibodies specific for SYCP3 (ab15093), RPA (ab87272), SUN1 (ab103021), H2A (ab18255), H2B (ab1790) and LAMIN B1 (ab16048) and mouse antibodies specific for SYCP3 (ab97672), γH2AX (ab26350) and TRF1 (ab10579) were purchased from Abcam. A rabbit antibody specific for DMC1 (sc-22768) was purchased from Santa Cruz Biotechnology. Rabbit antibodies specific for TRF2 (NB110-57130) and SYCP1 (NB300-229) were purchased from Novus Biologicals (Lihhleton, CO, USA). Mouse antibodies specific for α-tubulin (T9026) and FLAG (F1804) were purchased from Sigma-Aldrich. The rabbit antibody specific for the DDDDK-tag (PM020) that was used for co-immunoprecipitation was purchased from Medical & Biological Laboratories (Nagoya, JP).
The generation of the antibody specific for SHCBP1L has been previously described (36 (link)). The anti-FBXO47 antibody recognizes the peptide corresponding to the amino acid sequence DLDLPGTKEETALLE-C of FBXO47.
The generation of the antibody specific for SHCBP1L has been previously described (36 (link)). The anti-FBXO47 antibody recognizes the peptide corresponding to the amino acid sequence DLDLPGTKEETALLE-C of FBXO47.
10
Assessing DNA Damage and Telomere Dysfunction
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To detect DNA damage and telomere dysfunction, the γH2AX foci and TIFs were analyzed 48 h after lipofection. In brief, human chondrocytes were seeded onto coverslips and cultured for 24 h at 37 °C in an incubator under 20% O2/5% CO2. After chondrocytes had been cultured for 24 h, pcDNA3.1-Sirt6, siRNA Sirt6, or corresponding controls were transfected by lipofection. Forty-eight hours after lipofection, chondrocytes were fixed for 10 min with 4% paraformaldehyde in PBS, followed by permeabilization with 0.25% Triton X-100 in PBS for 10 min. The cells were subsequently washed with PBS, blocked for 1 h with 1% BSA (Sigma-Aldrich) in PBS containing 0.1% Tween-20, and then incubated with an anti- TRF-1 mouse monoclonal antibody (TRF-78, ab10579, Abcam, 1:1000 dilution). γH2AX was detected by incubation with a rabbit polyclonal antibody (Phospho-Histone H2A.X Ser139, Cell Signaling, 1:100 dilution, #9718).
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