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Fig. 2 | Epigenetics & Chromatin

Fig. 2

From: Telomere-specific chromatin capture using a pyrrole–imidazole polyamide probe for the identification of proteins and non-coding RNAs

Fig. 2

Affinity purification of the telomeric chromatin from mouse erythrocyte leukemia (MEL) cells by PI-PRICh. a Preparation of a negative control probe, masked TH59-DB, for telomeric chromatin isolation. TH59-DB was mixed and incubated with 50 times excess double-stranded oligonucleotide (TTAGGG)4/(CCCTAA)4 (telomeric oligonucleotides). b Purification validation of the telomeric repeat DNA with TH59-DB and masked TH59-DB. Telomeric repeat-containing plasmids were purified with TH59-DB (lane 2, 3) but not masked TH59-DB (lane 4, 5). Each fraction (Input, Flow-through, Elution) was analyzed by agarose gel electrophoresis and EtBr staining. The positions of the telomeric repeat-containing plasmid and the empty vector are indicated. c Silver staining of proteins obtained from telomeric chromatin purification with TH59-DB or LNA probes for the telomere repeat. (left) Chromatin isolation was performed with masked TH59-DB and TH59-DB. Input representing 0.001% of the starting material (104 cells equivalent, lane 1), 8% of the masked TH59-DB pull-down fraction (lane 2) and of the TH59 pull-down fraction (lane 3). (right) Silver staining of proteins obtained from PICh with scrambled (LNA control, lane 5) or telomere LNA probes (LNA telomere, lane 6). d Enlarged images of the boxed region between 50 and 75 kDa in c to show their similar band patterns. e Western blot analysis for TRF1 in each fraction of PI-PRICh. Input representing 0.0005% of the chromatin extracts, 4% of the materials of masked TH59-DB pull-down fraction or TH59-DB pull-down fraction. f List of proteins detected by mass spectrometry analysis of the material purified by TH59-DB from MEL cells. The results from two independent experiments are shown. The top ten proteins are sorted by the total number of peptides

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