His Tag Aptamer

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Binding of the His-tag protein to an aptamer bound chromatographic resin. Ten mL of 50% agarose coupled streptavidin was placed in a 1.5 ml Eppendorf tube and washed with distilled water. Then it was washed three times with a BW buffer (17.5 g/L NaCl, 50 mM phosphate buffer NaH2P04/Na2HP04, 0.1% (v/v) Tween 20, pH 7.5) . Next, the resin was suspended in 300 µl of a BW buffer containing 20 mg of 5'- biotinylated either Bl or a reference aptamer with the (ACTG)xlO GAGGAAC ATGCGTCGCAAAC sequence. The samples were incubated at room temperature for lh. After incubation, the resin was washed three times with the BW, followed by two washes with an A S buffer (137 mM NaCl, 12.7 mM KC1, 10 mM Na2HP04, 2 mM KH2P04, 5 mM MgC12, 0.1 % (v/v) Tween 20, pH 7.4) . After the last washing, the resin with the bound aptamer was suspended in 300 µΐ of the A S buffer, and the recombinant human PCNA protein was added, to a final concentration of 0.13 mg/ml. In this experiment, two variants of the PCNA protein were used: a ) with His-tag and b ) without His-tag. The resin with the protein was incubated at room temperature for lh. After incubation it was washed four times with the A S buffer, suspended in a GLB buffer (50 mM Tris-HCl, 2% SDS (w/v) bromophenol blue 2% (w/v) , 10% glycerol , 200 mM β-mercaptoethanol , pH 6.8) and incubated for 5 min at 95 °C . The resulting sample was subjected to SDS-PAGE (Laemmli method)

5'-GTTTGCCGGTGGGCAGGTCTAGGGTCTGCTCGGGATTGCGGAGGAACATGCGTCGCAAAC-3 ' (SEQ ID. No. 1 ) , 5'-GTTTGCCGGTGGGCAGGTTTAGGGTCTGCTCGGGATTGCGGAGGAACATGCGTCGCAAAC-3 ' (SEQ ID. No. 2 ) ,

https://www.aptagen.com/aptamer-details/?id=163 5'dGpdCpdTpdApdTpdGpdGpdGpdTpdGpdGpdTpdCpdTpdGpdGpdTpdTpdGpdGpdGpdApdTpdTpdGpdGpdCpdCpdCpdCpdGpdGpdGpdApdGpdCpdTpdGpdGpdCp