Figure 2.

Elongation products generated upon incubation with immunoaffinity-purified recombinant human telomerase. (A) Silver stain of an SDS-PAGE gel containing samples loaded as follows: 2.5 μl of rabbit reticulocyte lysate containing hTER and FLAG-tagged hTERT (RRL, lane 1), 10 μl of immunopurified recombinant telomerase washed with buffer containing 0.3 M NaCl (α-FLAG 0.3M, lane 2), or 0.6 M NaCl (α-FLAG 0.6M, lane 3). The protein enriched by immunopurification is indicated to the left with an arrow. Duplicate samples were electrophoresed on an SDS-PAGE gel, transferred to a membrane, and probed with anti-hTERT antibody. The order of samples is as follows: 2.5 μl of rabbit reticulocyte lysate containing no DNA (-ve, lane 1) or hTER and FLAG-tagged hTERT (RRL, lane 2), 10 μl of immunopurified recombinant telomerase washed with buffer containing 0.3 M NaCl (α-FLAG 0.3M, lane 3), or 0.6 M NaCl (α-FLAG 0.6 M, lane 4). The molecular mass (in kilodaltons) of protein markers is indicated at right. A longer exposure was performed to allow detection of hTERT synthesized in rabbit reticulocyte lysates (bottom; see lane 2). (B) Standard elongation assays were conducted with one of the following: 20 μl of partially purified telomerase from Raji cells (Raji DEAE, lanes 2, 3, 5, and 6), 20 μl of rabbit reticulocyte lysate containing hTER and FLAG-tagged hTERT (RRL lysate, lanes 8, 9, 11, and 12), 10 μl of immunopurified recombinant telomerase washed with buffer containing 0.3 M NaCl (α-FLAG 0.3M, lanes 14, 15, 17, and 18), or 0.6 M NaCl (α-FLAG 0.6M, lanes 20, 21, 23, and 24). Primer sequence is indicated above the corresponding lane: telomeric DNA is represented in black and nontelomeric DNA in white. The products resulting from incubation with (T₂AG₃)₂ are shown in lanes 2, 3, 8, 9, 14, 15, 20, and 21. The products arising from incubation with (T₂AG₃)₂X₁₂ are shown in lanes 5, 6, 11, 12, 17, 18, 23, and 24. See Table 1 for oligonucleotide sequence. The products were resolved on a 12% polyacrylamide sequencing gel. The ³²P-5′ end-labeled input oligonucleotides are shown to the left of the corresponding telomerase elongation reactions (I; lanes 1, 4, 7, 10, 13, 16, 19, and 22). Pretreatment with RNase A is indicated (+) for the samples corresponding to lanes 3, 6, 9, 12, 15, 18, 21, and 24.