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Penetratins 41
2. Add 10 to 50 molar ratio of reactive FITC dye to the peptide solution.
3. Incubate for 2 h at 4°C with gentle agitation.
4. Add 100 mM glycine (final concentration) and incubate for 2 h at 4°C
(glycine deactivates free FITC). Purify the peptide by dialysis (spectrapor
membranes, cut off 1000 d, Spectrum) against PBS at 4°C overnight with
at least four changes. Purify the labeled AntpHD by gel filtration
(microspin columns bio gel p6, Biorad) or by dialysis (spectrapor membranes,
cut off 3000 d, Spectrum) against PBS at 4°C overnight with at
least four changes.
2.5.1.1.2 Biotinylation Postsynthesis
Solutions
1. 100 mM sodium bicarbonate buffer, pH 8.5: dissolve 1.7 g NaHCO3 in
180 ml distilled water. Adjust to pH 8.5 with NaOH and complete to 200
ml with distilled water.
2. 1 M glycine: dissolve 7.5 g in 80 ml distilled water. Adjust to 100 ml with
distilled water.
3. Sulfo-NHS-biotin (MW 443.42).
Steps
1. Dilute 1 mg peptide in 1 ml 50 mM sodium bicarbonate buffer, pH 8.5
(final concentration).
2. Dissolve a 5-M ratio of Sulfo-NHS-biotin (biotin/peptide) directly in the
tube (alternatively, Sulfo-NHS-biotin can be predissolved in bicarbonate
buffer just before use).
3. Incubate for 2 h at 4°C.
4. Add 100 mM glycine (final concentration) and incubate for 2 h at 4°C
(glycine deactivates free Sulfo-NHS-biotin).
2.5.1.2 Internalization of AntpHD or Penetratin Peptides
Solutions
1. Culture medium.
2. Ethanol/acetic acid (9:1): mix 90 ml ethanol with 10 ml acetic acid. Store
at –20°C. Do not store more than 1 week.
3. PBS 10% NCS (or FCS): dilute 10 ml of 10 × PBS (Life Technologies)
in 80 ml distilled water. Add 10 ml NCS.
4. PBS 500 mM NaCl: dilute 10 ml of 10 × PBS (Life Technologies) and
10 ml 5 M NaCl in 100 ml distilled water.
Only for internalization of coupled peptide:
5. 1 M MgCl 2: dissolve 20.3 g MgCl 2. 6H 2O in 80 ml distilled water. Adjust
to 100 ml with distilled water.
6. 10× deoxyribonuclease I (300 µg/ml): dissolve 1 mg DNase I in 3.2 ml PBS.
Add 67 µl of 1 M MgCl 2. Filter, sterilize, and store at –20°C in aliquots.