Editing Synthesis of NLS Peptide

High-purity (&gt;95%) S413-PV peptide: ALWKTLLKKVLKAPKKKRKVC)<br> 

reverse NLS peptide (ALWKTLLKKVLKAVKRKKKPC)<br> 

and scrambled sequence (KTLK-VAKWLKKAKPLRKLVKC)<br> 

<br> 

peptides were modified with an acetyl group at the N-terminus<br>and had an amide group at the C-terminus. Peptides were tagged<br>with Nanogold (Monomaleimido Nanogold, Nanoprobes, NY,<br>d 1.4 nm) by forming a covalent bond between the thiol group<br>of peptide and the maleimide group of label by incubation at a<br>molar ratio 2.5:1 in 50% methanol at 30 °C for 90 min.<br>Methanol was removed and the conjugate concentrated by<br>speed-vac to reach 50-100 ÎŒM concentration of labeled peptide.<br>The conjugate with 1:1 peptide/Nanogold ratio was purified to<br>homogeneity by size-exclusion chromatography on Superdex<br>Peptide HR10/30 in buffer containing 35 mM Tris-HCl pH 7.5,<br>105 mM NaCl, and 30% (v/v) acetonitrile or by reversed phase<br>chromatography on Pro RPC HR5/2 (Pharmacia, Sweden) in<br>water-acetonitrile gradient in the presence of 0.1% TFA and<br>concentrated again. 

<br>Treatment of Cells with Peptides. Cells were seeded onto<br>glass coverslips in 24-well plates, grown to 80-90% confluence,<br>and incubated with culture medium containing 0.1-3 ÎŒM gold-<br>labeled peptides at 37 °C for 10 min to 24 h depending on the<br>experimental setup. After incubation, the cells were washed<br>twice with cell culture medium and processed for electron<br>microscopy (see below). For low-temperature experiments, cells<br>were preincubated in culture medium at 10 °C for 30 min,<br>followed by treatment with peptides at the same temperature<br>for 1 h.<br><br><br> 

[[Category:Transfection]]