Protein Analysis FAQ
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.
