C.B.S. Scientific HTLE-7002 User Manual

Page 16

C. B. S. Scientific 16

HTLE-7002

Then put the plate on top of it, cellulose
side up (Fig. D). Make a small cross
mark (+) where the sample and the
marker dye origins should be using a
very soft-leaded pencil. Note that the
position of the origin and dye spots will
vary depending on the electrophoresis
buffer used e.g., to the left side for pH
1.9 and 4.72 buffers and in the middle
for pH 8.9 buffer. Make short hatch
marks on the sides of the plate adjacent
to the sample and dye origin marks (see
stencil outline). At the very top of the
plate, we write an identification number
which refers to a specific sample.

Place the plate in a rack while additional
plates are prepared. The one shown in
upper middle of picture (Fig. E) is made
from stainless steel, will hold up to 10
plates and can be used to store and
transport plates without damaging their
surface.

Sample and Marker Dye Application

To apply the sample to the thin layer cellulose plate the following things are required: I) an area on the lab bench with a
flat surface, shown here is a thick glass plate (Fig. D), ii) a microfuge to centrifuge the samples, iii) an air hose (equipped
with a Nalgene 0.2

μm nitrocellulose filter unit to trap aerosols and particulate matter) or an air blower to dry down the

samples as they are applied and iv) Gilson P10 micropipetors with drawn out tips to apply the samples. Also needed
is marker dye, composed of a mixture of 5mg/ml

ε-DNP-lysine (yellow) and 1ml.mg xylene cyanol FF (blue) solubilized

in water containing 30-50% (v/v) of pH4.72 buffer. The dye mixture, which looks green in solution, will separate
during electrophoresis into its yellow and blue components indicating that electrophoresis is complete. The exact
position of the DNP-lysine marker relative to the origin is useful in determining the position of an electrically neutral
zone. The same marker dye is also used to determine the extent of migration during the chromatography steps (see
Stencil Outline for TLC Plates in Appendix, Page 36).