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abYsis which allows most of our antibody analysis to be performed is still available at www.abysis.org

What to I need to get my protein ready for Crystallography

This is a list of FAQs compiled by Dr. Kimberly Watson about how to prepare a protein for crystallography trials.

How much protein do I need?

10mg is sufficient for initial crystallization trials. Restricted trials are also possible with 2-5mg. Ideally you want as much as you can so that protein used for crystallization trials is also available for the actual crystals. (A different prep. might need slightly different conditions.)

What concentration should the protein solution be?

10-15mg/ml is typical, but crystals have been grown using 5-70mg/ml. Absolute minimum is 1mg/ml. If you know that your protein aggregates at 10mg/ml, then there is no point in producing a solution of this strength!

How pure must the preparation be?

Quite simply as pure as you can get it! Ideally >98% and at least >90%. Purities as low as 80% have been known to produce crystals, but your chances are much lower. Make use of CD spectra, dynamic light scattering (DLS), gel filtration and EM techniques to check for aggregation, denaturation, etc.

What tags and trimmings should I use?

There is no simple answer. Use what works, try everything!

What buffer should I use?

Ideally water so as not to interfere with conditions used for crystallization trials. However, most proteins have poor solubility in pure water. Avoid high salt concentrations - around 20mM is typically OK (certainly less than 100mM), but this depends on the salt type. TRIS is the best choice. Avoid phosphate and ammonium sulphate if possible as these can cause problems.

What pH should I use?

pH4-9 is typical, but start at the pI of the protein. This is one of the parameters that will be varied during crystallization trials.

Where can I get more information?

The Hampton Research web site has lots of useful information on crystallization and preparing samples for crystallography.