DIY Cryoconcentration Device
by Vijay Shankar, Labtimes 01/2014
Cryoconcentration is traditionally used to produce ice cider by freezing out apple juice. The ice moves to the top of the barrel, while the high-sugar juice remains at the bottom and may be fermented. A similar process takes place during freeze-centrifugation of proteins.
Protein solutions are quite often too diluted to be detected by SDS-PAGE. A newly developed micro-scale freeze-centrifugation protocol allows their fast and easy concentration.
In 2012, a Mexican group reported a simple protein concentration method based on freeze-centrifuging (see Lab Times, 4/2012). Recently, the team presented a refined protocol of their method enabling the rapid and simple micro-scale concentration of proteins prior to SDS-PAGE (Virgen-Ortiz et al., Anal. Biochem. ePub. 09 Sep 2013).
In industry, proteins are concentrated in large volumes for further analysis via freezing and centrifugation (freeze-dialysis concentration). The principle is that proteins and other solutes keep the frozen fraction in a slushy state, from which the proteins can be separated by centrifugation. Virgen-Ortiz and colleagues have basically downsized this method from large scale to the microtube level and come up with a clever, albeit simple homemade cryoconcentration device.
All you need for micro-scale freeze-centrifugation is a freezer set up at -70°C, 1.5 ml Eppendorf-tubes, 0.2 ml PCR microtubes, a cooled centrifuge and, of course, your protein fractions. To construct the cryoconcentration device, drill a hole in the lid of a 1.5 ml microtube with a diameter of 6 mm or use a hot pin with the same diameter. Next, cut the bottom off a PCR tube, turn it upside down with the lid tightly closed, add 200 µl of your diluted protein fraction and let it freeze at -70 °C for 30 minutes, upside-down. Then insert the PCR tube in the hole of the 1.5 ml tube and spin the assembled cryoconcentrator at 6000 rpm and 4 °C for 5 minutes. The centrifugal force brings down the concentrated protein solution into the 1.5 ml tube, while the ice is captured in the 0.2 ml PCR tube.
The Mexican team tested their freezecentrifugation technique on four commercial proteins, namely horseradish peroxidase, soybean trypsin inhibitor, horse myoglobin and fructosyltransferase. To this end, diluted protein fractions from gel filtration chromatography were concentrated by the freeze-centrifugation technique described above and visualised on SDS-PAGE. According to the authors, the cryo-concentrated proteins are easily visible on the gel, in contrast to the diluted samples.
That’s all fine, however, there is a fly in the ointment: the authors have used only three commercial reference enzymes, which are usually abundant, to prove their method. The fructosyltransferase preparation, brought about from gel filtration chromatography, is also at a relatively pure state.
So, there is no clue to how well the freeze-concentration technique works for proteins in intermediary purity stages. The definite litmus test would be to apply the method in a real lab setting, i.e. during the purification of low-abundant proteins. Considering the cheap, simple and rapid protocol, however, this could be well worth a try.
Last Changed: 05.02.2014