Yingying.lu at 22:24, 15 November 2011

2011-11-15T22:24:54Z

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High-purity (>95%) S413-PV peptide: ALWKTLLKKVLKAPKKKRKVC) 

reverse NLS peptide (ALWKTLLKKVLKAVKRKKKPC) 

and scrambled sequence (KTLK-VAKWLKKAKPLRKLVKC) 

 

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

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

[[Category:Transfection]]

Synthesis of NLS Peptide - Revision history

Yingying.lu at 22:24, 15 November 2011