I am planning to crystallize an antibody Fab fragment. I have several crystallization screening buffer sets in the laboratory, including popular sets sold by Hampton Research (eg. Index, Crystal Screen etc.). However I do not have screens designed for antibodies crystallization like Hampton Research Low Ionic Strength Screen.
Is it better to buy or prepare the buffers like those included in the Low Ionic Strength Screen? Did antibodies you have crystallized readily form crystals in conditions found with the common protein screens as well?
Michal, microseeding in *random* screens works incredibly well with Fab frags. Out of 70 structures solved by a biotech co in 4 yrs, 38 benefited from microseeding, either in getting new hits or better crystals (ref 2 below). It also allows you to control the number of crystals in each well by diluting the seed stock.
Refs:
[1] 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
[2] Microseed Matrix Screening Crystallization of Antibody Fragments
and Antibody-Antigen Complexes. RAMC, Strasbourg, France, 2011.
Galina Obmolova, Biologics Research, Centocor R&D
http://hamptonresearch.com/documents/ramc/RAMC2011_T11_Obmolova.pdf
[3] Further information on the theory and practice of the MMS method
is available at the Douglas Instruments web-site,
http://www.douglas.co.uk/mms.htm and http://www.douglas.co.uk/MMS_proc.htm
[4] Patrick Shaw Stewart, Stefan Kolek, Richard Briggs, Naomi Chayen,
and Peter Baldock. Random Microseeding: A Theoretical and Practical
Exploration of Seed Stability and Seeding Techniques for Successful
Protein Crystallization.
http://pubs.acs.org/doi/abs/10.1021/cg2001442
It is advised to buy the kit, as it is more useful. The wise option is; there are many Fab fragment crystals have been solved and str. are been deposited in PDB . You can find these conditions and prepare the buffer accordingly. I believe if your protein is only Fab fragment at least one in 20 published conditions may give some positive results. However, i do suggest to have screen as each preparation is different.
That all depends on how much protein you have, and if you have robots at your disposal (small drops). But I would set up the screens at hand, and start the thinking/ordering afterwards.
Do try different temperatures. And if nothing happens after a reasonable time, put the plate in the fridge or at 37.
If you get any crystal-like objects, set up the same screen(s) using these objects as a seed as recommended above.
Consider adding the antigen if available and crystallize the complex. At least for nanobodies, solo crystallization doesn't work that well (but that might be due to the on-average-long CDR3).
Maybe do a quick check whether the protein actually likes being in low ionic strength in the first place.
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