4 Qualities to Evaluate Prior to Beginning Protein Crystallization
One of the most important factors for achieving successful crystallization is the
quality of
your protein. Optimizing a protein's purity and homogeneity, while maintaining its
composition and structure will increase your chances for successful crystallization.
Prior to crystallization, these qualities should be evaluated:
Purity
Protein purity is
critical for successful crystallization.
We typically recommend a purity of at least 95% as assessed by SDS-PAGE and Coomassie-blue staining.
A
general
metal-based purification can result in a protein that is more than 95% pure.
Posttranslational modifications should be homogenous throughout the sample.
Homogeneity
The protein should exist throughout the sample in the same form and size.
Use gel filtration to ensure a homogenous sample following purification.
When possible, evaluate sample using dynamic light scattering to confirm one
homogenous protein population.
Composition
Proteins degrade throughout the purification process.
Therefore, it is recommended to set up your crystallization so it
immediately
follows purification.
Structure
It's a good idea to run circular dichroism (CD) spectroscopy prior to
crystallization to confirm the protein's secondary structure.
If possible, check the protein's bioactivity with an appropriate
quantitative
assay.
