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Or gP al en ai sc e&d Bo i on mo to al ed cj uu ls at r mC ha re gmi ni ss try
DOI: 10.1039/C8OB00742J
Journal Name
ARTICLE
Conjugation processes
Conflicts of interest
Conjugation of model proteins and fluorescent linkers. In the There are no conflicts to declare.
C162S
case of p38
60 µM protein and 120 µM linker 10 were
mixed and incubated for 10 minutes at room temperature in
the dark in PBS buffer (pH 7.4). As the second step, the Acknowledgements
appropriate dye (11–15) was added in 240 µM concentration
Help of Gábor Glatz with SDS-PAGE gel imaging is greatly
acknowledged. Present work was supported by the Hungarian
Scientific Research Fund (OTKA-NN-116265), the “Lendület”
Programme of the Hungarian Academy of Sciences (LP2013-
for 10 min, too. The excess reagents were removed by using
SpinPrep column (Sigma, St Louis, MO, USA) filled with
Sephadex G-25 “Fine” desalting gel (Pharmacia Fine Chemicals,
Sweden). In the case of MK2 60 µM protein and 120 µM linker
5
5/2013) and the National Research, Development and
10 were mixed and incubated for 10 minutes at room
Innovation Office, Hungary (VEKOP-2.3.3.-15-20016-00011).
temperature in the dark in PBS buffer (pH 7.4) completed with
additional 160 mM NaCl, 0.1% IGEPAL and 400 µM TCEP. As
the second step the dye 12 was added. The excess reagents
were removed by using SpinPrep column filled with Sephadex
G-25 “Fine” desalting gel.
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FRET measurements
2
C162S
The labeled p38
samples were prepared in 1.0 µM
concentration in PBS (pH 7.4) buffer. The labeled MK2 peptide
and protein samples were prepared in 16, 8, 4, 2, 1, 0.5, 0.25,
0.125, 0.0625, 0.03125 µM concentrations for the titration.
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fluorescence signal (λ : 420 nm; λem: 450-700 nm) by using a
ex
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6
071–6078.
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