214
Consequently, the methods presented below may be useful for the
structure solution of other Ca 2+ binding proteins where the more
traditional HA methods fail.- Prepare a solution containing 60 mM SmAc in the crystalliza-
tion solution. - Pipette 2–3 μL of this solution onto clean well of a 24-well
crystallization tray. - Open a crystallization well containing the crystals and collect
a single crystal using a cryoloop and transfer to the heavy atom
condition. - Cover the well for 2 min before transferring the crystal briefl y
to the cryo-solution and freezing at 100 K ( see above).
Fluorogenic substrate assays are used to assess the activity of active
TbMCA2 and associated mutants, by measuring the release of a
fl uorophore (e.g., AMC) from the hydrolysis of a fl uorescent pep-
tide by the enzyme. All assays should be run in technical triplicate
and include a negative control (containing no protein). In addi-
tion, all replicates and controls should be included on the same
96-well plate. The amount of each reagent is given for a standard
assay, but the volume of protein should be optimized as required.- Exchange the protein into the Exchange Buffer using a PD-10
desalting column. - Concentrate the protein using a centrifugal concentrator, or
dilute as necessary, to around 0.5–1 mg/mL. The protein can
be stored at 4 °C but for best results it should be used as soon
as possible after purifi cation ( see Note 17 ). - Dilute the fl uorogenic substrates to a 100 μM working stock
in ddH 2 O. Prepare enough for 20 μL per well. - Immediately prior to running the assay, dilute protein to
between 0.2 and 0.4 mg/mL in 1× Assay buffer. This is a 40×
working solution requiring 5 μL per well and giving a fi nal
assay concentration of 5–10 μg/mL. - Place a 96-well black bottomed plate on ice and add the
reagents in the following order:
(a) ddH 2 O to give a fi nal volume of 200 μL
(b) 100 μL 2× Assay Buffer
(c) 5 μL protein (or buffer for a negative control). This vol-
ume can be adjusted as necessary.
(d) 20 μL substrate - Remove the plate from the ice, place in the fl uorescence plate
reader, and equilibrate to ambient temperature (20–25 °C) for
5 min.
3.5 Activity Assays
3.5.1 Fluorogenic
Substrate Activity Assays
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