11. SAP-97 monoclonal antibody (cat. No. 75-030; NeuroMab,
Davis, USA).
12. SAP-102 polyclonal antibody (cat. No. 124202; Synaptic Systems, Goettingen, Germany).
13. Horseradish peroxidase (HRP) secondary antibody.
2.5 Human Blood
Plasma Stability Assay
1. Compound (2.5 mM stock solution in water).
2. Human blood plasma, pooled (3H Biomedical, Uppsala,
Sweden).
3. 5% (w/v) aqueous trichloroacetic acid.
2.6 In Vitro Toxicity
Experiment (MTT
Assay)
1. Compound
2. MK-801 (Tocris Bioscience, Bristol, UK)
3. 5 mM sodium azide in Hank’s Balanced Salt Solution (HBSS)
or acetic acid
4. N-methyl-D-aspartic acid (NMDA)
5. 5 mg/mL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) in PBS buffer.
2.7 Equipment
1. Agilent 6410 Triple Quadrupole Mass Spectrometer with electron spray ionization (ESI) coupled to an Agilent 1200 HPLC
system (ESI-LC/MS), equipped with C18 reverse phase columns: Zorbax Eclipse XBD-C18, 4.6 mm  50 mm for the
analysis of peptide and Agilent Poroshell 300SB-C18,
2.1 mm  75 mm for the characterization of protein (Agilent
Technologies, Santa Clara, USA)
2. Waters ACQUITY system for analytical reverse phase
ultrahigh-performance liquid chromatography (RP-UPLC)
with a C18 reverse phase column (ACQUITY UPLC BEH
C18 1.7 μm, 2.1 mm  50 mm) for the analysis of peptides
and a C8 column (ACQUITY UPLC BEH C8 1.7 μm,
2.1 mm  50 mm) for the analysis of proteins (Waters Corp.,
Milford, USA)
3. Waters 2545Q system for preparative reverse phase highperformance liquid chromatography (RP-HPLC) (Waters
Corp., Milford, USA) with a C18 reverse phase column Zorbax
300 SB-C18, 21.2 mm  250 mm (Agilent Technologies,
Santa Clara, USA) for the purification of peptides and a reverse
phase C4 column 250 Â 10 mm (Jupiter®, Phenomenex,
Torrance, USA) for the purification of proteins.
4. A ¨ KTA explorer 100 FPLC system (GE Healthcare, Chicago,
USA), equipped with HiLoad® 16/600 Superdex® 75 pg
column (GE Healthcare, Chicago, USA) for size-exclusion
chromatography (SEC)
162
Dominik J. Essig et al.
Davis, USA).
12. SAP-102 polyclonal antibody (cat. No. 124202; Synaptic Systems, Goettingen, Germany).
13. Horseradish peroxidase (HRP) secondary antibody.
2.5 Human Blood
Plasma Stability Assay
1. Compound (2.5 mM stock solution in water).
2. Human blood plasma, pooled (3H Biomedical, Uppsala,
Sweden).
3. 5% (w/v) aqueous trichloroacetic acid.
2.6 In Vitro Toxicity
Experiment (MTT
Assay)
1. Compound
2. MK-801 (Tocris Bioscience, Bristol, UK)
3. 5 mM sodium azide in Hank’s Balanced Salt Solution (HBSS)
or acetic acid
4. N-methyl-D-aspartic acid (NMDA)
5. 5 mg/mL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) in PBS buffer.
2.7 Equipment
1. Agilent 6410 Triple Quadrupole Mass Spectrometer with electron spray ionization (ESI) coupled to an Agilent 1200 HPLC
system (ESI-LC/MS), equipped with C18 reverse phase columns: Zorbax Eclipse XBD-C18, 4.6 mm  50 mm for the
analysis of peptide and Agilent Poroshell 300SB-C18,
2.1 mm  75 mm for the characterization of protein (Agilent
Technologies, Santa Clara, USA)
2. Waters ACQUITY system for analytical reverse phase
ultrahigh-performance liquid chromatography (RP-UPLC)
with a C18 reverse phase column (ACQUITY UPLC BEH
C18 1.7 μm, 2.1 mm  50 mm) for the analysis of peptides
and a C8 column (ACQUITY UPLC BEH C8 1.7 μm,
2.1 mm  50 mm) for the analysis of proteins (Waters Corp.,
Milford, USA)
3. Waters 2545Q system for preparative reverse phase highperformance liquid chromatography (RP-HPLC) (Waters
Corp., Milford, USA) with a C18 reverse phase column Zorbax
300 SB-C18, 21.2 mm  250 mm (Agilent Technologies,
Santa Clara, USA) for the purification of peptides and a reverse
phase C4 column 250 Â 10 mm (Jupiter®, Phenomenex,
Torrance, USA) for the purification of proteins.
4. A ¨ KTA explorer 100 FPLC system (GE Healthcare, Chicago,
USA), equipped with HiLoad® 16/600 Superdex® 75 pg
column (GE Healthcare, Chicago, USA) for size-exclusion
chromatography (SEC)
162
Dominik J. Essig et al.