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May 26, 2016

How to improve cold room protein purification

By Abhijit Parkhe, Product Manager

Are your proteins unstable at room temperature and need to be purified in a cold room?

Here are some tips for how to improve chromatography system and column performance when working with our products at low temperature.

Key factors that impact protein purification at low temperature

There are two main reasons for performing protein purification at low temperatures: preserve the integrity of proteins and samples as well as reduce proteolytic activity and prevent unwanted degradation of the target protein.

Whatever the reason is, there are many factors to consider and several easy steps that can improve the outcome. Here I will focus on the three main components of a cold room protein purification experiment: buffers and samples, chromatography columns, and systems. All of them can make a significant difference to the quality of results.

1. Buffers and samples at low temperature

Pay special attention to preparing your buffers and samples in advance. Before starting, refrigerate buffers together with the system, chromatography resin, or column for 12 hours to stabilize the temperature. Once the temperature has stabilized, check buffer pH and adjust it if needed, especially for Tris buffers. Refrigerate your sample, especially if it has not been prepared at low temperature.

2. Chromatography columns at low temperature

Resin and column type determine how chromatography columns are used at low temperature. Here are some tips on how to improve column performance in cold rooms:

  • Remember that buffer viscosity increases at lower temperatures. So the maximum flow rate will be lower, but column hardware pressure limits will not change compared to when working at room temperature. 
  • For some affinity resins, binding of proteins can be affected by low temperature. In those cases you need to lower the flow rate during sample application. 
  • For size exclusion chromatography (gel filtration) columns, you might need to reduce the flow rate by 50%.

Columns are precious consumables. Here are few things to keep in mind to protect them:

  • To protect a packed bed from collapsing, maintain flow rates within the maximum flow rate limits recommended. Ensure that pressure drop over the packed bed is always within the prescribed limits. 
  • For safety, maintain column hardware pressure within recommended limit. 
  • Reduce flow rates when working with viscous fluids.

3. Chromatography systems at low temperature

Some simple precautions greatly impact results when running lab-scale ÄKTA systems at low temperatures:

  • Bring your system to the desired temperature 12 h or more before starting your experiment. 
  • Keep your system switched on when the cold room is on to avoid condensation and potential equipment damage. 
  • Switch off the system when the cold room is switched off. 
  • If a cold cabinet is used, keep it open to avoid overheating. 
  • Calibrate pressure offset when all buffers and sample have reached the desired temperature. Refer to system user documentation for details. 
  • Check fittings, tubing connections, and other connections as soon as the system is moved between different temperature environments. 
  • Use a low-temperature compatible computer or place the computer outside the cold room. 
  • ÄKTA avant, ÄKTAprime plus, and ÄKTA pure perform well in cold environments enabling use of high-viscosity buffers needing lower flow rates (< 0.5 mL/min). 
  • Running size exclusion chromatography columns on an ÄKTA start system at low temperature with high-viscosity buffers can be difficult. To complete the run, you might need to bring the instrument and column to room temperature. 
  • High back pressures are more likely to occur with HiPrep Sephacryl columns than with HiTrap columns.

Get more guidance on protein purification at low temperatures in a cue card format for your lab.