Macromolecular Crystallization Facility

(photo: introductory image for the site)

Generator

PROTOCOLS

Protein Preparation- Minimum Requirements

We know that pure protein samples increase the likelihood of successful crystallization, and that the worst contaminants are those that are closely related to the target protein (i.e. degradation products). For this reason, we ask you to provide us with as pure a protein sample as possible, as judged by SDS-PAGE.

Typical starting concentrations for crystallization trials are in the 10-15 mg/mL range, and sample volumes will depend on the number of screens you would like to set up. We request approximately 55uL of sample per 96-well screen, preferably in water or a low ionic strength buffer. Please avoid using phosphate buffer.

For more information about sample preparation, do not hesitate to contact us!

Crystallization Trials

Once you submit your sample, we can start our crystallization trials with one or all of our Qiagen 96-well crystallization screens, and these include:

The JCSG+ Suite- a sparse matrix screen we use for initial investigations

The PACT Suite- for a systematic analysis of the effect of pH, anions, and cations

The AmSO4, PEGs and MPDs Suites- 96-well screens using different precipitant types

Our 96-well plates have room for three samples per well, therefore we can screen from one to three different protein samples per plate (or 3 different concentrations of the same protein), allowing us to test from 96 to 288 conditions per trial.

Submitting Your Protein Sample

Please be prepared to supply us with basic information regarding your sample. We would like to know:

the name and origin of your protein (i.e. is it from E. coli?),

expected molecular weight,

theoretical pI,

amino acid composition, and anything else outlined in the checklist provided.

A lot of this information can be obtained by simply entering your protein's name into the Expasy proteomics server or Expasy's ProtParam Tool.

Please make note of these details when filling out the Sample Submission Form.

A Note On Sample Purity

What methods did you use to purify your protein? If you used a fusion-tag to ease in purification, did you remove it? If you are hoping to crystallize a complex of proteins, did you run it through a size-exlusion column first? These are things we need to think about (or discuss in-person, if you prefer) before we attempt to crystallize your sample. Make note of all of this in your Sample Submission Form.

Please provide us with a picture of your protein at its purest form, as judged by SDS-PAGE.

Sample Stability and Homogeneity

There are many different ways to learn about the stability of your protein, and to find out if your sample is monodisperse. You won't want to set up crystallization trials if your protein sample has degraded, aggregated or come out of solution over time.

Techniques such as gel filtration, dynamic light scattering and analytical centrifugation can be used to determine whether your protein sample contains aggregates, while simple analysis by SDS-PAGE, circular dichroism and differential scanning calorimetry can be used to check for degradation and thermal stability.

Click here to view our simple protein stability test protocol.

Also from this web page:

Buffer Selection

Less Is More!

While the ideal buffer for your protein sample is water, many proteins won't tolerate this lack of ionic strength in solution. If this is the case for your sample, we recommend that you take a "less is more" approach to selecting the appropriate buffer. This simply means that you should use the lowest possible amount of buffer (i.e. 5-10mM), salts or glycerol to keep your sample in solution.

Even a little can sometimes be too much...

We discourage the use of phosphate buffers and addition of most salts to your final sample buffer, as small molecules such as these can crystallize on their own and can therefore be misleading.