Freezing Samples

The following is our suggested strategy for snap freezing biological samples. We have tested this method many times, and it seems to work well for most samples.

We minimise the degradation of our protein samples by ensuring that each sample goes through only one freeze/thaw cycle, and by ensuring that the freezing process is as rapid as possible, reducing the chance of the formation of crystalline ice. 

Freezing and thawing protein samples almost certainly result in some degradation of the protein sample, however, for long-term storage of purified protein there aren’t many other feasible options.  We recommend snap freezing small aliquots of concentrated protein in individual thin-walled PCR tubes. The PCR tubes, in turn, are stored in a labelled centrifuge tube. We suggest using a 15 mL centrifuge tube for storing 10 or fewer PCR tubes; a 50 mL centrifuge tube will hold up to 40 PCR tubes.

Concentrated protein samples generally freeze more successfully than dilute samples. We do not recommend adding glycerol to samples before snap freezing.

The damage to the sample may come about through a number of mechanisms: ice formation and the related step increase in protein concentration, cold denaturation, pH changes, and viscosity changes, to name a few.  Protein tends to denature on surfaces – freezing a sample may introduce crystalline ice into the sample, thus providing a large surface area onto which protein molecules can adsorb and denature.

Here is a link to a Nature Methods publication (Murphy et al) that outlines some of the potential problems.

You will need:

  1. Styrofoam box with lid (for dry ice)
  2. Open Dewar (for liquid nitrogen)
  3. Styrofoam box with ice for protein sample
  4. 15 or 50 mL centrifuge tube for storage
  5. 0.2 ml PCR tubes or strips
  6. Labels
  7. Dry ice
  8. Liquid nitrogen
  9. Tweezers

Take care to wear appropriate gloves (either blue cryogen gloves or white cotton gloves underneath nitrile gloves).  Always wear safety glasses or goggles, and remember that dry ice and liquid nitrogen will burn quickly and painlessly.

Determine that you know where the final storage place is for your protein – This might change how you label your samples

For each sample, prepare an appropriate (15 mL or 50 mL) centrifuge tube by labelling with your name, sample name, and date  (use a permanent marker). You can try to label the PCR tubes, but most labels don’t stick well, and permanent markers rub off. Simple labels on the lid of flat-lidded PCR are probably the most reliable.

Place some dry ice (2-5 cm depth) in the Styrofoam box with a lid, and place the labelled centrifuge tube(s) in the dry ice to pre-chill.

Aliquot your sample into the labelled thin-walled PCR tubes which are sitting on ice.  Aliquot 100 µl or less of protein into each tube, to ensure rapid freezing.

Add liquid nitrogen to the Dewar or other appropriate container to a depth of 1-2 cm.

Using the tweezers, pick up a tube or a strip, and quickly plunge it into the liquid nitrogen.  Try to keep the tube or strip upright as you plunge, so the protein sample stays in the bottom of the PCR tube.

Move the PCR tubes containing frozen protein into the appropriate pre-chilled, labelled centrifuge tube.

Dispose of the residual liquid nitrogen and dry ice appropriately (e.g. pour into the sandbox in the X-ray room). Do not pour liquid nitrogen onto the floor or down the sink.