fractions are too small to be measured reliably in a plate reader
with full area collection plates and offer too little volume for
further analytical steps. Maintain fraction volumes of at least
100 μL to obtain separations with a sufficient number of steps
and with enough volume for further analysis. Conversely, in the
case of small number of steps, do not select them in a fashion
giving fraction volumes greater than the volumetric capacity of
a collection plate (typically ~0.3 mL for most 96 well full area
microplates) as this will cause the plates to flood and will lead to
cross contamination of samples and robot contamination.
10. To measure fractions in the UV spectrum, use UV transparent
plates (Corning Inc., NY, USA).
11. To determine fraction volumes, also measure each collected
fraction at 900 and 990 nm and determine volumes, and then
pathlength (by dividing the volume by the cross sectional
surface area of the wells in a collection plate), as described
in [15].
12. When preparing buffers ensure to prepare them at a volume
that includes at least 150 μL of excess for 96-well deep square
well plates (Fisher Scientific, Loughborough, UK) and an
additional of 150 μL for the aliquoting of the buffers in plates
to blank the measured fractions.
13. When defining gradients in the HT scale, define them on a
basis of mM CV
À1 or %B CV
À1 and not as mM or %B per unit
time, since, when scaling down or up using RoboColumns this
takes place on a constant residence time basis. In this case, the
flowrate is adjusted so as to match the residence time, as
opposed to the linear velocity that is typically implemented
with conventional chromatography.
14. Include at least ~500 μL of excess to account for dead volumes
in the wells of 48-well deep well square plates (Elkay Ltd.,
Hampshire, UK).
15. Residence times of up to 4 min and 12 min for the 0.2 mL and
0.6 mL RoboColumns respectively are possible with the
provided liquid classes; On EVO robots it is not possible to
implement different liquid classes per channel and hence
RoboColumn screening experiments are grouped based on
residence times.
16. A probe capable of fitting into wells of 96-well plates needs to
be used (~4 mm shaft diameter).
HTPD Purification of Viral Antigens
133
with full area collection plates and offer too little volume for
further analytical steps. Maintain fraction volumes of at least
100 μL to obtain separations with a sufficient number of steps
and with enough volume for further analysis. Conversely, in the
case of small number of steps, do not select them in a fashion
giving fraction volumes greater than the volumetric capacity of
a collection plate (typically ~0.3 mL for most 96 well full area
microplates) as this will cause the plates to flood and will lead to
cross contamination of samples and robot contamination.
10. To measure fractions in the UV spectrum, use UV transparent
plates (Corning Inc., NY, USA).
11. To determine fraction volumes, also measure each collected
fraction at 900 and 990 nm and determine volumes, and then
pathlength (by dividing the volume by the cross sectional
surface area of the wells in a collection plate), as described
in [15].
12. When preparing buffers ensure to prepare them at a volume
that includes at least 150 μL of excess for 96-well deep square
well plates (Fisher Scientific, Loughborough, UK) and an
additional of 150 μL for the aliquoting of the buffers in plates
to blank the measured fractions.
13. When defining gradients in the HT scale, define them on a
basis of mM CV
À1 or %B CV
À1 and not as mM or %B per unit
time, since, when scaling down or up using RoboColumns this
takes place on a constant residence time basis. In this case, the
flowrate is adjusted so as to match the residence time, as
opposed to the linear velocity that is typically implemented
with conventional chromatography.
14. Include at least ~500 μL of excess to account for dead volumes
in the wells of 48-well deep well square plates (Elkay Ltd.,
Hampshire, UK).
15. Residence times of up to 4 min and 12 min for the 0.2 mL and
0.6 mL RoboColumns respectively are possible with the
provided liquid classes; On EVO robots it is not possible to
implement different liquid classes per channel and hence
RoboColumn screening experiments are grouped based on
residence times.
16. A probe capable of fitting into wells of 96-well plates needs to
be used (~4 mm shaft diameter).
HTPD Purification of Viral Antigens
133