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Freezing Samples

The following is our suggested strategy for snap freezing biological samples. We have tested this method many times, but be warned that all samples behave differently.

Freezing and thawing protein samples almost certainly results in some degradation of the protein sample, however, for long term storage of purified protein there aren’t many other feasible options.  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.

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.

You will need:

  1. Styrofoam box with lid (for dry ice)
  2. Styrofoam box (for liquid nitrogen)
  3. Styrofoam box with ice for protein sample
  4. Storage Box: SSI 5630-T4 IsoFreeze® block (Interpath)
  5. 0.2 ml PCR tubes or strips
  6. Labels
  7. Aluminium freezing block
  8. Dry ice
  9. Liquid nitrogen
  10. Tweezers

Left to Right: IsoFreeze block, Styrofoam box & lid and Aluminium block & PCR strip

Take care to wear appropriate gloves (either the 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 – make sure that there is room for the PCR rack in the -80 freezer before you start.

Place some dry ice (2-5 cm depth) in the Styrofoam box with lid.

Label both the lid and the side of the freezer block with labels suitable for -80ºC (the barcode labels from C3 are appropriate). Place the PCR rack and lid in the box with the dry ice.

Label up individual PCR tubes or PCR strips. It is recommended that you put 100 ul or less of protein into each tube, to ensure rapid freezing. Note that concentrated protein samples generally freeze more successfully than dilute samples (100 uL would be sufficient for four 96-well crystallisation trials).

Place the labelled PCR tubes / strips in the ice bucket, and fill each with 100 mL of concentrated protein solution. Close the lids of the PCR tubes or press the strip lid on the strip of tubes (we use PCR tubes here as they are thin walled, and allow for the most rapid freezing of the sample).

Check that the aluminium block is dry, place it in the remaining empty Styrofoam box, and pour liquid nitrogen over it to cover by 1-2 cm.  Wait for a couple of minutes until the block comes down to liquid nitrogen temperature (the nitrogen will stop boiling).  The block should be completely submerged in the liquid nitrogen, with just a couple of millimetres of liquid nitrogen over the block.

Using the tweezers, pick up a tube or a strip, and quickly place it in the aluminium block.  Ensure that there is no ice or water adhering to the outside of the tube before you plunge it into the nitrogen.  Continue until all the tubes are sitting in the block.

Keeping the PCR rack on the dry ice, quickly move the frozen PCR tubes into the pre-chilled freezer block, place the lid on the freezer block and put the lid back on the Styrofoam box.

If you’re doing this on site: Carry the foam box with dry ice and the freezer block to a -80ºC freezer. Place the freezer block in the -80ºC freezer.

If you’re sending the samples to C3 via courier: Cover the IsoFreeze block in dry ice in a robust Styrofoam box and secure the lid using packing tape. Print a label with our address and secure this to the TOP of the lid. Make sure to label the parcel as PERISHABLE and make sure that your courier service is able to deliver the package within a suitable time frame.

Dispose of the residual liquid nitrogen appropriately (pour into the sandbox in the X-ray room). Do not pour liquid nitrogen onto the floor or down the sink.  The aluminium block will take a long time to warm up to room temperature – make sure that it will not be picked up by someone whilst it is coming up to room temperature.