Quality Control Strategies for Membrane Protein Purification Before Cryo-EM
A Practical Guide with Insights from Dr. Melanie McDowell from the Max Planck Institute of Biophysics
Introduction
If you're a structural biologist working on membrane proteins, you already know: sample quality is everything. Before you even think about freezing grids or booking cryo-EM time, the real work happens at the bench—optimizing expression, solubilization, and purification conditions to get a stable, homogeneous, functional sample.
This guide is for you.
It’s built around real questions from researchers like you—scientists pushing the boundaries of what’s possible in cryo-EM. During a recent webinar on quality control for membrane protein purification, participants raised common but critical challenges. In response, Dr. Melanie McDowell, Research Group Leader at the Max Planck Institute of Biophysics and an expert in membrane protein structural biology, shared detailed, experience-based advice.
From detergent screening and nanodisc reconstitution to buffer optimization and sample stability, Dr. McDowell’s answers are grounded in what works—and what doesn’t—when preparing samples for cryo-EM. Her lab routinely uses tools like nanoDSF and DLS to assess stability and homogeneity, ensuring that only the best-behaved samples make it to the microscope.
Whether you’re troubleshooting aggregation, testing new solubilization strategies, or deciding when (and how) to switch detergents, this guide offers practical solutions you can apply directly—especially if you’re using or considering technologies like Prometheus Panta to support your workflow.
Key Takeaways from the Q&A: Practical Advice from the Lab
1. Exploring Detergent Alternatives: Amphipols & Nanodiscs
Some cryo-EM structures can be solved without detergents at all. Dr. McDowell shared successful cases using amphipols and MSP-based nanodiscs, which provided better particle distribution in the ice.
Tip: When detergents compromise protein behavior or grid preparation, test detergent-free systems early. Amphipols and nanodiscs can offer enhanced stability and behaviour more like a soluble protein.
References: https://doi.org/10.1016/j.molcel.2020.08.012
https://doi.org/10.1038/s41467-023-42867-2
2. Endogenous Expression Systems: A Viable but Selective Approach
One researcher asked about purifying membrane proteins directly from endogenous levels via cryo-lysis. Dr. McDowell confirmed this is feasible—in their case from fungi such as Chaetomium thermophilum—but they often tag the genomic copy to enable purification. For that organism, bead beating was more effective than cryo-lysis.
Tip: Choose lysis methods based on your organism and protein’s stability. The feasibility depends very much on the expression level of the protein/complex; several thousand copies per cell as estimated by https://www.yeastgenome.org can be enough. Screen for expression levels and optimize accordingly.
3. Starting With DDM: Proceed With Caution
Is DDM safe for solubilization if you're planning to switch detergents later? Dr. McDowell advised testing a complete purification in DDM first. Although DDM is relatively mild, it can stabilize less favorable conformations and switching might not reverse this.
Tip: Avoid detergent switching unless necessary. If you must, monitor stability closely at every step using nanoDSF or DLS.
4. Nanodisc Assembly and Protein Loss: Experience Varies
A participant mentioned losing 70–80% of protein during nanodisc reconstitution. Dr. McDowell noted that while loss can occur, the magnitude depends heavily on the system. Her lab often sees heterogeneous SEC profiles, with a portion of protein forming aggregates or oversized assemblies.
Tip: Replace Bio-Beads with α-cyclodextrin for detergent removal. It reduces precipitation and gives more reproducible results.
5. SMALPs and Copolymers: A Moving Target
Asked about the reliability of SMALPs, Dr. McDowell explained that earlier SMA polymers were challenging—leading to heterogeneous samples that got stuck on columns. Newer copolymers are more promising, but success depends on matching the right polymer to your system.
Tip: Be prepared to screen a variety of copolymers. Tag choice and purification strategy may need to be adapted.
Note: For strategies on copolymers screening, check this application note.
6. Lysis Techniques: Tailor to Your System
Cell disruption must be efficient yet gentle and temperature-controlled. Dr. McDowell uses a microfluidizer for E. coli and bead beating or cryo-milling for fungi. She emphasized the importance of the right speed for low-speed centrifugation to avoid prematurely pelleting target membranes.
Tip: Small tweaks in lysis and pelleting protocols can dramatically affect yield and quality.
7. Concentrating Amphipol-Solubilized Proteins: Choose the Right Filter
Some attendees noted protein sticking during concentration of amphipol-solubilized proteins. While Dr. McDowell and her team did not face this issue yet, she recommended testing different concentrators to find the one that fits best with membrane proteins.
Tip: When in doubt, compare membrane materials between filter brands—it can save your sample.
8. Micelle Size Variability: Understand the Context
Concentration-dependent changes in protein-detergent complex size were reported. McDowell has observed this too, but emphasized it’s usually minor. Aggregation, not micelle size shift, is often the bigger issue.
Tip: Use nanoDSF and DLS at your target working concentration to confirm sample homogeneity before cryo-EM.
9. Mass Photometry in Detergents: Know the Limits
Mass photometry can be challenging with detergents due to micelle interference. Dr. McDowell noted it works best when the protein is significantly larger than the micelle and when using tight-binding detergents like LMNG.
Tip: Use it for larger complexes and well-optimized detergent systems. Consider detergent dilution or removal before analysis if needed.
10. Copolymer Extraction vs. Nanodisc Reconstitution
SMA/DIBMA extraction can be efficient, but her early experiences showed purification limitations (e.g., filter clogging, heterogeneity). Nanodiscs can also yield heterogeneous samples—but these are more amenable to purification via SEC.
Moreover, extraction efficiency with copolymers depends very much on the type of membrane and the type of copolymer. The recent development of a much wider variety of copolymers will hopefully make it easier to find one that is suitable for your system.
Tip: If particle distribution is an issue for cryo-EM, nanodisc reconstitution followed by SEC often gives more defined particles, even if starting yields are lower. Yet, the new range of available copolymers will open new approaches.
11. Effective Detergent Exchange for Screening
To ensure detergent exchange during detergent screening, Dr. McDowell adds test detergents at their solubilization concentration and dilutes the protein sample with detergent-free buffer to dilute the detergent used for the initial purification (often DDM). Behavior changes observed via nanoDSF/DLS confirm the swap is effective.
Tip: Always include controls (e.g., the purification detergent) and use screening tools to assess protein behavior across detergents.
12. pH and Detergent Sensitivity: Pay Attention During Concentration
Stability issues are often concentration-dependent. A protein may tolerate a suboptimal pH when dilute, but aggregate during concentration if not in ideal conditions.
Tip: Shift to optimal pH or buffer conditions before concentration to avoid loss due to aggregation. For example, elute at pH 7.5 and immediately drop pH to 6.0 before concentrating.
Conclusion: Small Changes, Big Impact
As Dr. Melanie McDowell’s Q&A responses revealed, membrane protein purification for cryo-EM is full of nuance. Success often hinges on subtle but crucial decisions—many of which can be guided by biophysical characterization tools like Prometheus Panta.
By adopting rigorous quality control strategies throughout the workflow – especially during purification, detergent screening, and concentration—structural biologists can increase their chances of generating high-resolution structures and avoid costly setbacks.
Explore more:
- Learn about Prometheus Panta for protein stability and quality control
- Watch the full webinar recording
- Get more resources on Membrane Proteins
