Two detailed protocols on how to prepare a microseed stock can be found here:
“Procedure for making the microseed stock” from Patrick D. Shaw Stewart (Douglas Instruments Ltd).
“Matrix Seeding” from Janet Newman (Collaborative Crystallisation Centre (C3)).
The preparation of the microseed stock will be carried out by the researcher. PCC will assist in the preparation of the microseed stock by providing “Seed Beads”(Hampton Research, HR2-320) and a “Micro-Tools Set”.
The initial steps for setting up a microseeding experiment is identical to the vapor diffusion crystallization experiment described in the section Experimental Setup.
The addition of the microseed stock to each drop of the vapor diffusion crystallization experiment is carried out by the Gryphon-LCP at 20°C or 4°C. The researcher specifies the volume of the microseed stock to be added to the three crystallization wells. The microseed stock volume added is commonly in the range between 20nl and 40nl.
The volume for the microseed stock required for setting up a crystallization plate can be found in Sample Volume.
The Oxford Protein Production Facility-UK provides an Automated Microseed Protocol for Protein Crystallization
The following list of microseeding papers is an introduction to the microseeding crystallization technique:
list of papers
Allan D’Arcy, et al.
Microseed matrix screening for optimization in protein crystallization: what have we learned?; Acta Crystallogr F Struct Biol Commun. 2014 Sep;70(Pt 9):1117-26.
Allan D’Arcy, et al.
An automated microseed matrix-screening method for protein crystallization;
Acta Crystallogr D Biol Crystallogr. 2007 Apr;63(Pt 4):550-4.
Marisa Till, et al.
Improving the success rate of protein crystallization by random microseed matrix screening.n; J Vis Exp. 2013 Aug 31;(78).
Gregory C.Ireton, et al.
Microseed matrix screening to improve crystals of yeast cytosine deaminase;
Acta Cryst. (2004). D60, 601-605
Galina Obmolova, et al.
Promoting crystallization of antibody–antigen complexes via microseed matrix screening; Acta Cryst. (2010). D66, 927–933
Armando G. Villasenor, et al.
Acoustic matrix microseeding: improving protein crystal growth with minimal chemical bias; Acta Cryst. (2010). D66, 568–576
Patrick D. Shaw Stewart, et al.
Random Microseeding: A Theoretical and Practical Exploration of Seed Stability and Seeding Techniques for Successful Protein Crystallization;
Cryst. Growth Des. 2011, 11, 3432–3441
Abuhammad Areej, et al.
Structure of arylamine N-acetyltransferase from Mycobacterium tuberculosis ..."; Acta Crystallographica Section D: Biological Crystallography 69, no. 8 (2013): 1433-1446
Enrico A. Stura, et al.
Epitaxial jumps; Journal of crystal growth 196, no. 2 (1999): 250-260.
Stefan A. Kolek, et al.
A novel microseeding method for the crystallization of membrane proteins in lipidic cubic phase;
Acta Crystallographica Section F, Volume 72; Part 4; April 2016; Pages 307-312.
see also: RAMC2011_T11_Obmolova (PDF, 1 MB)