May 04, 2026
A Practical Workflow for Protein Concentration and Buffer Exchange Using Ultrafiltration for Phase Separation Studies
- Jianxiong Chen1
- 1Department of Pathology, The Tenth Affiliated Hospital, Southern Medical University (Dongguan People's Hospital), Dongguan,Guangdong, 523059, China.
- Data Analysis Protocol
Protocol Citation: Jianxiong Chen 2026. A Practical Workflow for Protein Concentration and Buffer Exchange Using Ultrafiltration for Phase Separation Studies. protocols.io https://dx.doi.org/10.17504/protocols.io.3byl4pobolo5/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: May 04, 2026
Last Modified: May 04, 2026
Protocol Integer ID: 316341
Keywords: protein phase separation, studying protein phase separation, ultrafiltration for phase separation study, protein solubility, individual proteins during storage, phase separation study, protein concentration, centrifugal ultrafiltration, practical workflow for protein concentration, using ultrafiltration, phase separation, protein sample, membrane drying, separation assay, nucleolar proteins sensitive to physicochemical environment, affinity purification, preparation of nacl, protein, following affinity purification, individual protein, prevention of membrane drying, nacl concentration, exchange of recombinant protein, nta chromatography, disordered protein, recombinant protein, nucleolar protein
Abstract
This protocol describes a streamlined workflow for the concentration and buffer exchange of recombinant proteins following affinity purification, optimized for downstream in vitro phase separation assays. Proteins purified by Ni-NTA chromatography are subjected to centrifugal ultrafiltration to remove imidazole and exchange into a defined basal buffer. To preserve protein solubility and structural integrity, NaCl concentrations are empirically optimized for individual proteins during storage. Prior to phase separation assays, protein samples are adjusted to controlled buffer conditions, enabling systematic evaluation of phase behavior under varying ionic strength and macromolecular crowding conditions. The protocol further outlines the preparation of NaCl and polyethylene glycol (PEG) gradients and the setup of small-volume reactions for microscopic observation. Critical parameters, including centrifugation conditions, prevention of membrane drying, and accurate accounting of background salt contributions, are discussed. This method provides a robust and reproducible approach for studying protein phase separation and is broadly applicable to intrinsically disordered proteins and nucleolar proteins sensitive to physicochemical environments.
Purpose
This protocol describes the concentration and buffer exchange of recombinant proteins after Ni-NTA purification using centrifugal ultrafiltration devices. The procedure removes imidazole and excess salts and prepares proteins in a defined low-salt buffer suitable for in vitro phase separation assays under varying NaCl and PEG conditions.
Materials
- Ultrafiltration devices (0.5 mL, 10 kDa MWCO, e.g., Amicon‱ Ultra-0.5)
- Refrigerated centrifuge (capable of 10,000–14,000 × g)
- Ice bath
- Pipettes and low-retention tips
- Liquid nitrogen
- −80 °C freezer
Buffers
Basal Buffer (Low-salt buffer)
- 10 mM Tris-HCl, pH 7.5
- 2 mM DTT
- 150 mM NaCl
Proteins were maintained in 10 mM Tris-HCl (pH 7.5), 2 mM DTT, and 150 mM NaCl prior to phase separation assays.
Used for buffer exchange and as base condition for phase separation assays.
The NaCl concentration in the storage buffer was optimized individually for each protein to maintain solubility and stability.
Ultrafiltration Device Pretreatment
Add 0.5 mL ultrapure water to the filter unit.
Incubate on ice for 2–5 min.
Centrifuge at 14,000 × g, 4 °C for 5 min.
Discard filtrate.
Protein Loading and Initial Concentration
Pre-cool centrifuge to 4 °C.
Add up to 0.5 mL protein solution (Ni-NTA elution) to the filter unit.
Centrifuge at 10,000–14,000 × g, 4 °C for 3–5 min.
Concentrate to ~100 μL retentate.
Buffer Exchange (Repeat 3 Times)
Add 300 μL basal buffer, gently mix.
Centrifuge at 10,000–14,000 × g, 4 °C for 3–5 min.
Repeat this wash step three times to remove imidazole and adjust buffer composition.
Final concentration step:
Centrifuge for 5–15 min until ~50–80 μL remains.
Protein Recovery
Invert the filter device into a clean collection tube.
Centrifuge at 1,000 × g, 4 °C for 2 min.
Collect concentrated protein.
Storage
Measure protein concentration immediately.
Aliquot samples.
Snap-freeze in liquid nitrogen.
Store at −80 °C.
Preparation for Phase Separation Assay
Experimental Variables
- PEG6000: 0%, 2%, 5%
- NaCl: 75–300 mM gradient
- Protein concentration: defined range depending on experimental design
- Final reaction volume: 10 μL
Stock Solutions
- PEG6000 stock: 20% and/or 50% (w/v)
- NaCl stock: 2 M
- Base buffer: 10 mM Tris-HCl, 2 mM DTT
Reaction Setup
Mix protein, PEG, NaCl, and buffer to desired final concentrations.
Gently pipette to mix (avoid bubbles).
Incubate briefly at room temperature.
Observe under microscope:
DIC imaging
Fluorescence channel (if applicable)
Notes and Critical Considerations
Protein Quality and Buffer Effects
- Ni-NTA eluates typically contain imidazole, which can interfere with phase separation; thorough buffer exchange is essential.
- Maintain proteins in a low-salt basal buffer before applying NaCl gradients to ensure accurate control of ionic strength.
- Avoid using buffers containing glycerol or detergents unless required.
Centrifugation and Concentration
- Use low acceleration/deceleration settings to prevent protein aggregation.
- Do not centrifuge to dryness; always retain a minimal volume (~50 μL).
- Monitor for precipitation or turbidity, which may indicate instability.
Handling of Ultrafiltration Devices
- Avoid touching the membrane with pipette tips.
- Protein adsorption to the membrane may occur, especially at low concentrations.
- Reuse of filters is not recommended for critical experiments due to contamination risks.
DTT Stability
- DTT should be freshly added to buffers before use.
- Avoid long-term storage of DTT-containing buffers.
Phase Separation Assay Design
- Ensure that NaCl contribution from protein stock is considered when calculating final concentrations.
- PEG and NaCl stocks should be prepared in compatible buffers.
- Work quickly to minimize protein degradation and oxidation.
Storage and Freeze–Thaw
- Avoid repeated freeze–thaw cycles.
- Aliquot into small volumes for single use.