Biomedical Engineering Reference
In-Depth Information
Equilibrate a second chromatographic chamber with Solvent 2
as described above. The dried sheet is turned to 90
◦
and developed
for the second dimension until the solvent front reaches about
5 mm below the top.
Drythesheetagainandkeeponthevaporofconcentrated
HCl.Identifytheredspotsincomparisonwithachromatogramof
DABTH amino acid test mixture and determine migration relative
to the bluish spots of the markers (schematic drawing of a 2
×
2-cm
TLC plate is given in Fig. 3.1).
References
Findlay JBC, Geisow MJ (eds.) (1989) Protein sequencing: a practical ap-
proach. IRL Press, Oxford, p 123
Choli-Papadopoulou T, Skendros Y, Katsani K (1997) In: Kamp RM, Choli-
Papadopoulou T, Wittmann-Liebold B (eds.) Protein structure analy-
sis. Springer, Berlin, p 137
3.1.2 Thin-Layer Chromatography of Nucleoside Phosphates
The following solvents are suitable for chromatography of adeno-
sine triphosphate (ATP) and derivatives:
0.2 M ammonium hydrogencarbonate in H
2
O
Solvents
or
0.4 M formic acid in H
2
O
or
0.2 M sodium hydrogenphosphate in H
2
O
Use a sheet of polyethyleneimine cellulose (PEI Cellulose), 50
×
200 mm, for chromatography. Mark the start about 15 mm away of
the shorter side with a pencil. Apply the sample with a pipet or
syringe. If larger volumes have to be used, apply them in several
portions and/or a short line.
Develop the chromatogram ascending in a chamber lined with
filter paper and equilibrated with one of the solvents.
After drying the sheet with a cold fan
32
P-labeled nucleotides
are identified by autoradiography or a PhosphoImager. Unlabeled
substances are identified by fluorescence quenching (see below).
The most effective solvent is sodium hydrogenphosphate, if the
detection is not done with phosphate reagent (e.g., Hanes' reagent;
see below).
3.1.3 Gradient Thin-Layer Chromatography of Nucleotides
1 .7M ammonium formiate in H
2
O
Solvents
1
2 M ammonium formiate in H
2
O
1
3 M ammonium formiate in H
2
O
2 .2M lithium chloride in H
2
O
2
1 M lithium chloride in H
2
O
2
1.6 M lithium chloride in H
2
O
