a consistent final screening concentration of DMSO, wells are
backfilled, where necessary, with the appropriate volume of DMSO.
All ARPs have specific wells assigned for assay controls, and the
positioning of these control compounds is dependent on the assay
format used (see Note 8). Control compounds allow analysis of
both inter- and intra-plate variation, calculation of Z
0 values [13],
and for setting the normalization window.
The assay protocol used is usually a 10 μL enzyme mixture
addition followed by a 10 μL substrate addition and finally a
30 μL acid step to stop the assay. Of course, while these volumes
in 384-well plates bring a disadvantage in that they result in large
assay volumes (50 μL), which increases assay reagent costs, they are
required to achieve efficient firing (see Note 9).
There are many variables that appear to influence firing from
these plates and hence the quality of the data produced. The
meniscus formed by various dispensers or whether plates are centrifuged can cause problems with firing. Use of a Multidrop™
Combi, or similar dispenser, on a slow or medium speed for the
final addition step of stop reagent to the assay is recommended.
This reduces bubble formation in the well and appears to produce
more consistent liquid surfaces. Centrifugation after both the
enzyme and substrate additions is possible if the final addition
step is added with a Multidrop™ Combi dispenser. Assay plates
should always be stored at room temperature as using cold plates
reduces effective firing.
An advantage of AMI-MS technology is the ability to read an
assay while it is still progressing. For example, measurement of realtime assay progression can be obtained by adding enzyme and
substrate to a plate and then repeatedly reading on the AMI-MS
to obtain a significantly larger volume of kinetic data.
3.4 Automated
Screening
3.4.1 Automation
of Assay Plate Production
The simple nature of these assays makes automation for production
of assay plates straightforward. The combination of three dispensers and a robot arm to transfer plates between dispensers at the
appropriate time has successfully been applied to automate this
process for a range of assays. When running AMI-MS at high
throughput, the assay should be stopped to ensure that incubation
times are consistent, usually using an acid such as formic acid or
acetic acid. This enables the read to take place any time after the
assay is performed, and often the plates can be re-read over a week
after plate production, providing there is no breakdown of products
occurring (see Note 10).
3.4.2 Automation
of Assay Plate Read
on the AMI-MS
The Access™ Dual Robot System (Labcyte) will allow incorporation of both assay plate production and plate read into one automated system, as it allows the movement of plates from the plate
stacker to the read position. This will provide flexibility of running
224
Helen Plant et al.
backfilled, where necessary, with the appropriate volume of DMSO.
All ARPs have specific wells assigned for assay controls, and the
positioning of these control compounds is dependent on the assay
format used (see Note 8). Control compounds allow analysis of
both inter- and intra-plate variation, calculation of Z
0 values [13],
and for setting the normalization window.
The assay protocol used is usually a 10 μL enzyme mixture
addition followed by a 10 μL substrate addition and finally a
30 μL acid step to stop the assay. Of course, while these volumes
in 384-well plates bring a disadvantage in that they result in large
assay volumes (50 μL), which increases assay reagent costs, they are
required to achieve efficient firing (see Note 9).
There are many variables that appear to influence firing from
these plates and hence the quality of the data produced. The
meniscus formed by various dispensers or whether plates are centrifuged can cause problems with firing. Use of a Multidrop™
Combi, or similar dispenser, on a slow or medium speed for the
final addition step of stop reagent to the assay is recommended.
This reduces bubble formation in the well and appears to produce
more consistent liquid surfaces. Centrifugation after both the
enzyme and substrate additions is possible if the final addition
step is added with a Multidrop™ Combi dispenser. Assay plates
should always be stored at room temperature as using cold plates
reduces effective firing.
An advantage of AMI-MS technology is the ability to read an
assay while it is still progressing. For example, measurement of realtime assay progression can be obtained by adding enzyme and
substrate to a plate and then repeatedly reading on the AMI-MS
to obtain a significantly larger volume of kinetic data.
3.4 Automated
Screening
3.4.1 Automation
of Assay Plate Production
The simple nature of these assays makes automation for production
of assay plates straightforward. The combination of three dispensers and a robot arm to transfer plates between dispensers at the
appropriate time has successfully been applied to automate this
process for a range of assays. When running AMI-MS at high
throughput, the assay should be stopped to ensure that incubation
times are consistent, usually using an acid such as formic acid or
acetic acid. This enables the read to take place any time after the
assay is performed, and often the plates can be re-read over a week
after plate production, providing there is no breakdown of products
occurring (see Note 10).
3.4.2 Automation
of Assay Plate Read
on the AMI-MS
The Access™ Dual Robot System (Labcyte) will allow incorporation of both assay plate production and plate read into one automated system, as it allows the movement of plates from the plate
stacker to the read position. This will provide flexibility of running
224
Helen Plant et al.