crc press - E-Lib FK UWKS

Penetratins 43
see Reference 31). Coupling requires the presence of an SH group at either end of
the oligonucleotide. Modified oligonucleotides are available commercially with a
thiol or disulfide pyridinium group.
Solutions
1. 5 × SDS sample buffer: to make 20 ml, add 0.97 g Tris, 2 g SDS, 10 ml
glycerol (87%), and 0.5% bromophenol blue. Dissolve in distilled water
and adjust to pH 6.8.
2. 5 × reducing SDS sample buffer: 20 ml 5 × SDS sample buffer containing
1.5 g DTT.
Steps
1. Resuspend separately the oligonucleotide and the vector in distilled water
(1 to 10 mg/ml).
2. Quickly add an equimolar amount of vector to the oligonucleotide (after
resuspension oligonucleotides can form homodimers; even if this reaction
is relatively slow it is preferable to add the vector rapidly). Occasionally,
a small precipitate may appear. If so, the sample can be incubated at 65°C
for 15 min and, if the precipitate persists, 1 M NaCl or 20% DMSO (final
concentration) can be added.
3. Incubate for 2 h at 37°C.
4. Keep a 5-µg aliquot for testing the yield of the reaction and store the
remaining aliquots at –80°C.
5. Dilute 5 µg of the coupled product in 1 × SDS sample buffer (final
concentration). Run the sample on 20% SDS-PAGE with no reducing
agent. The same amount of coupled product diluted in 1 × reducing SDS
sample buffer or vector alone can be run as control.
6. Stain the gel with Coomassie blue. The yield of the reaction is routinely
above 50%.
2.5.2.1.2 Peptide
Basically, the coupling procedure is identical to the coupling with an oligonucleotide
except that TCEP is preferred to DTT as the reducing agent. The cargo peptide must
be ordered or synthesized with a disulfide pyridinium group or a cysteine at one
end, except if a cysteine is already in the peptide sequence. If this is the case, the
vector will react with the internal cysteine. 43 This does not affect the capacity of
internalization.
Solutions
1. TCEP (MW 286.65): resuspend TCEP in distilled water at the appropriate
concentration.
Steps
1. Resuspend separately the vector and the peptide in distilled and degazed
water (1 to 10 mg/ml). If the peptide is not hydrosoluble, it can be resuspended
in an appropriate mixture of water and DMSO or water and
dimethylformamide without interference with the coupling reaction.