212
- Measure protein absorbance at 280 nm ( A 280 ) using a spectro-
photometer and use this and the extinction coeffi cient
(M −1 cm −1 ) ( see Note 16 ) to calculate the approximate protein
concentration. - Concentrate the protein to around 7 mg/mL using a centrifu-
gal concentrator. The protein can be stored at 4 °C but for
best results it should be used immediately after purifi cation
( see Note 17 ). - Prior to crystallization, centrifuge the sample at 12,000 × g for
5 min in a benchtop centrifuge to remove any precipitates. - Pipette 500 μL of the Crystallization Solution into the reser-
voir of a 24-well sitting drop plate. - Without crystallization seeds: Pipette 3 μL of the purifi ed pro-
tein into the crystallization well, followed by 3 μL of the res-
ervoir solution. - With crystallization seeds: Pipette 3 μL of the purifi ed protein
into the crystallization well, followed by 1 μL of the seed stock
and 2 μL of the reservoir solution. - Seal the tray and place at 4 °C for around 3 weeks after which
time they can be moved to ~21 °C for storage and crystal
manipulation. - Check trays weekly for crystals.
- Pipette 50 μL of the Crystallization Solution into a microcen-
trifuge tube ( see Note 18 ). - Collect crystals from a crystallization drop using an appropri-
ately sized cryoloop. - Place the cryoloop into the solution and shake it to displace
the crystals into the solution. - After crystal collection, transfer the entire solution into a micro-
centrifuge tube containing a seed bead and vortex for 90 s. - Add a further 450 μL of the Crystallization Solution and vor-
tex for a further 90 s. - Prepare fi ve serial dilutions of this stock solution using the
Crystallization Solution e.g., 1:2, 1:10, 1:100, 1:500, 1:1,000
and store seeds in small aliquots at −80 °C or −20 °C. - Set up crystallization trays varying the seed stocks and moni-
tor the results. - Open the crystallization well containing the crystals and
pipette 4 μL of the reservoir solution onto a glass coverslip. - Add 1 μL of 100 % MPD and mix by pipetting gently up and down
(this results in the cryo-solution, which contains 20 % MPD) - Collect a single crystal from the crystal tray using an appropri-
ately sized cryoloop and transfer to the cryo-solution.
3.4.1 Crystallization
of Inactive TbMCA2 C213A
3.4.2 Preparation
of Seed Stocks
3.4.3 Cryoprotection
of TbMCA2 C213A Crystals
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