Procedure for making the microseed stock

 

·         Any crystalline protein material can be used for microseeding, including fine needles, “spherulites”, microcrystals, and irregular poorly-formed crystals. 

·         Note that the microcrystals in the seed stock are not stable because the seed stock contains very little protein.  Therefore the seed stock should be kept on ice and frozen as soon as possible, preferably at -80ºC.

·         If you have plenty of crystals, use the Seed Bead from Hampton Research, HR2-320, see http://hamptonresearch.com . 

·         If you have only a few small crystals, modify the procedure below by crushing the crystals with a probe and making a smaller volume of seed stock (e.g. 15 µl) without using the Seed Bead.

 

The method is based on the method of D’Arcy et al., adapted from Luft and DeTitta, references below.

 

 

1.       Put a Seed Bead (in its test-tube) into a bucket of ice to cool it.

2.       Open the well containing seed crystals, and crush the crystals with a probe to check that the crystals can be shattered (i.e. they are not cross-linked).

3.       Put 50 µl of the reservoir solution that gave the hit into the Seed Bead tube, keeping it on ice.

4.       Remove about 6 µl of reservoir solution from the tube, and transfer it to the well with crystals.  Dispense and suck back into the tip several times.  Change the volume setting on the pipette to e.g. 8 µl and transfer the mixture back to the Seed Bead tube.

5.       Wash the well with another 6 µl of solution from the Seed Bead tube, again transferring the washings back to the Seed Bead tube.

6.       Vortex the Seed Bead tube for two minutes.

7.       Use the original undiluted seed stock for MMS microseeding (seeding into a random screen).  For MMS not dilute 1:100 as instructed in the Hampton Research Seed Bead instructions.  The more crystals there are in your seed stock the more hits you will obtain.  Recommended volumes to use are 0.3 µl of protein, 0.2 µl of reservoir, and 0.1µl of seed stock for robotic work.  Before using the seed stock, agitate it in case the suspended crystals have settled in the tube.

8.       However, we suggest that you make a dilution series straight away, up to 1 in 1000.  Use these diluted seed stocks in later experiments if too many crystals are obtained.  Freeze all seed stocks immediately at -80ºC (or -20ºC if not available).

9.       MMS can be performed by hand (increase the volume) or with a robot that uses contact dispensing.

10.   Immediately after use, freeze the undiluted seed stock at -80ºC / -20 ºC.

 

 

References:

 

Luft, J. R. & DeTitta, G. T. (1999). ‘A method to produce microseed stock for use in the crystallization of biological macromolecules’.  Acta Cryst. D55, 988–993.

 

Allan D’Arcy, Frederic Villarda, May Marsh.  'An automated microseed matrix-screening method for protein crystallization'.  Acta Crystallographica section D63 (2007), 550–554.  On-line at http://scripts.iucr.org/cgi-bin/paper?S0907444907007652