12. When using confocal microscopy such as confocal, avoid
saturated images, which are not quantitatively useful, by
adjusting the saturation and gain settings.
13. Bleaching of a sample should be avoided.
14. TRICEPS coupling is favored under alkaline conditions and
the coupling buffer must not contain primary amines (e.g.,
Tris). The recommended buffer is 25 mM HEPES pH 8.2.
15. It is also recommended to use 50 μg of TriCEPS with a high
TriCEPS:ligand ratio to increase the number of carbohydrate
structures that can be captured per ligand; coupling ratio of
50 μg of TRICEPS per 100 μg of ligand is recommended.
16. The coupling reaction is competed by the hydrolysis of
NHS-ester and given that the hydrolysis occurs more readily
in dilute protein solutions, a more concentrated solution is
recommended. A coupling reaction with 100 μg of ligand per
50–100 μL of buffer is proposed.
17. The required ligand amount is roughly 100 μg per replicate
and the ligand buffer should not contain primary amines; in
case the ligand buffer is not compatible, the ligand should be
dialyzed against 25 mM HEPES pH 8.2.
18. Given that coupling of the cross-linker may affect the bioactivity or binding properties of the ligand, binding and functional
assays using the ligand-TriCEPS conjugates prior to the LRC
experiments are recommended.
19. During collection of cells, no reagents containing proteases can
be used as they will result in digestion of the extracellular
proteins.
20. A total number of approximately 10
7 cells per sample are
recommended; however, different cell types exhibit different
cell size and surface. As a rule of thumb, the cell pellet of each
sample at the end of the LRC should be between ca. 40 and
100 μL to render a sufficient protein concentration for further
analysis.
Acknowledgments
M.Z. and K.S.F. are supported by American Heart Association
Scientist Development Award 17SDG33411180, and by a grant
awarded under the Pitt Innovation Challenge (PinCh) through the
Clinical and Translational Science Institute of the University of
Pittsburgh ,
through
National
Institutes
of
Health,
UL1TR001857.
Disclosures: M.Z. along with Paul D. Robbins (Professor, University of Minnesota, Minnesota, MN, USA) hold a patent on the use
Validating Cardiac Targeting Peptide
111
saturated images, which are not quantitatively useful, by
adjusting the saturation and gain settings.
13. Bleaching of a sample should be avoided.
14. TRICEPS coupling is favored under alkaline conditions and
the coupling buffer must not contain primary amines (e.g.,
Tris). The recommended buffer is 25 mM HEPES pH 8.2.
15. It is also recommended to use 50 μg of TriCEPS with a high
TriCEPS:ligand ratio to increase the number of carbohydrate
structures that can be captured per ligand; coupling ratio of
50 μg of TRICEPS per 100 μg of ligand is recommended.
16. The coupling reaction is competed by the hydrolysis of
NHS-ester and given that the hydrolysis occurs more readily
in dilute protein solutions, a more concentrated solution is
recommended. A coupling reaction with 100 μg of ligand per
50–100 μL of buffer is proposed.
17. The required ligand amount is roughly 100 μg per replicate
and the ligand buffer should not contain primary amines; in
case the ligand buffer is not compatible, the ligand should be
dialyzed against 25 mM HEPES pH 8.2.
18. Given that coupling of the cross-linker may affect the bioactivity or binding properties of the ligand, binding and functional
assays using the ligand-TriCEPS conjugates prior to the LRC
experiments are recommended.
19. During collection of cells, no reagents containing proteases can
be used as they will result in digestion of the extracellular
proteins.
20. A total number of approximately 10
7 cells per sample are
recommended; however, different cell types exhibit different
cell size and surface. As a rule of thumb, the cell pellet of each
sample at the end of the LRC should be between ca. 40 and
100 μL to render a sufficient protein concentration for further
analysis.
Acknowledgments
M.Z. and K.S.F. are supported by American Heart Association
Scientist Development Award 17SDG33411180, and by a grant
awarded under the Pitt Innovation Challenge (PinCh) through the
Clinical and Translational Science Institute of the University of
Pittsburgh ,
through
National
Institutes
of
Health,
UL1TR001857.
Disclosures: M.Z. along with Paul D. Robbins (Professor, University of Minnesota, Minnesota, MN, USA) hold a patent on the use
Validating Cardiac Targeting Peptide
111