Home › Cell Biology › Microfluidic Chips for In Situ Crystal X-ray Diffraction and In Situ Dynamic Light Scattering for Serial Crystallography
Cell Biology JoVE (Open Access) Citable · DOI

Microfluidic Chips for In Situ Crystal X-ray Diffraction and In Situ Dynamic Light Scattering for Serial Crystallography

DOI: 10.3791/57133-v
What you'll learn
  • ✓ Fabricate microfluidic PDMS chips for in situ X-ray crystallography
  • ✓ Monitor protein crystallization using dynamic light scattering
  • ✓ Collect and process serial crystallography diffraction data at room temperature
Protocol

Biopharma Insights This protocol describes in detail how to fabricate and operate microfluidic devices for X-ray diffraction data collection at room temperature. Additionally, it describes how to monitor protein crystallization by dynamic light scattering and how to process and analyze obtained diffraction data.

Difficulty
advanced
Total time
~3–5 days (mold fabrication 1 day, chip preparation 1 day, crystallization screening 1–3 days, X-ray data collection 2–4 hours per sample)

Steps

1
Fabricate PDMS mold and in situ X-ray chip

Create polydimethylsiloxane (PDMS) master molds using photolithography or soft lithography, then replicate and bond PDMS to create microfluidic chips suitable for X-ray diffraction experiments.

▶ 00:38
2
Prepare fluid delivery ports and surface treatment

Establish inlet and outlet access ports on the microfluidic chip and apply surface treatments (e.g., coating) to optimize protein crystallization and sample flow characteristics.

▶ 04:59
3
Prepare protein samples and initiate crystallization

Concentrate and condition protein solutions, load samples into microfluidic channels, and set up crystallization conditions inside the microfluidic device.

▶ 06:51
4
Monitor crystallization via dynamic light scattering

Use in situ dynamic light scattering to track protein nucleation and crystal growth within the microfluidic chip in real time.

▶ 06:51
5
Collect and analyze X-ray diffraction data

Perform room-temperature in situ X-ray diffraction data collection on crystallized protein samples and process diffraction patterns for structural analysis.

▶ 08:51
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