One of the most widespread protein laboratory accessories are the MWCO (molecular weight cut-off) centrifugal filters which are commonly used for concentrating protein, as well as DNA. They are available commercially with different cut-offs including 3kDa, 30kDa, 50kDa, 100kDa, and so on. These little devices are expensive and hence demand proper usage and care to utilise their benefit to the full extent. Here, I am going to discuss some tips and tricks about their storage, use, and finally some troubleshooting information gathered from my personal experience with centrifugal filters.
Proper Use and Handling of Centrifugal Filters
Choosing the Right Size Cut-Off for Your Centrifugal Filters
As mentioned earlier, centrifugal filters are available commercially with different cut-offs. It is important to realize that MWCO filters are not meant for separating different molecular weight entities. If you want to separate molecules on the basis of molecular weight, you would need to use the well-established technique of size exclusion chromatography. MWCO filters can be used for buffer exchange and desalting. My starting point for the choice of cut-off is to start with a cut-off at least two times smaller than the molecular weight of the protein you want to concentrate.
However, if you use too small a cut-off, it will require lots of time and subsequent refillings. Moreover, if you use a small cut-off like a 3000 Da (3 kDa) cut-off for large protein size (let’s say 100kDa), there is possibility of concentrating low molecular weight proteins, which can potentially contribute to protein precipitation. On the contrary, if you use too high a cut-off, there will be more membrane surface which can provide more area for non-specific binding.
Reusing Used Centrifugal Filters
Because of the cost, it is compelling to use MWCO filters mulitple times. If you wish to do so, after each use, you should wash them with ultra-pure water, followed with 70% ethanol, and finally another wash with ultra-pure water. Once washed, it is imperative that proper storage conditions discussed in the following section are followed.
Although not ideal, MWCO filters can be reused if they don’t clog. There is generally drastic protein loss after concentrating or concentrating the protein starts to take unexpected long times if these filters are clogged. Despite this fact, they should be discarded after a maximum of three uses. These filters may start to leak after being used 2-3 times.
Make Sure You Store Your Used Centrifugal Filters Properly
When it comes to proper storage of used filters, the most fundamental thing to keep in mind is to never let the membrane dry. The trick is to keep the membrane of filters moist and cool after first use, otherwise the membrane “skin” cracks and then it is useless.
I’ve personally found adding PBS (1X) with little sodium azide (0.02% w/v) to be the best. Instead of this, you can also add 20% ethanol and then store it. Nevertheless, it is crucial to store the filter at 4°C (not in freezer or -20°C). The structure of those fragile polymers used to form the membrane are prone to damage by repeated freeze/thawing.
How to Use Your Centrifugal Filters
When starting to concentrate large volumes, I personally give an initial spin for 5 minutes to determine how fast the solution is flowing through the filter and then use that to decide the time required to spin. It is better to give a spin for no more than 15 minutes initially and then, using a pipette, gently mix the sample 2-3 times to avoid blocking the membrane to avoid losing your hard work and money at this stage. When mixing, you will see a more viscous protein solution close to the membrane swirl up and diffuse into the less concentrated solution. When pipetting, take special care not to touch and disrupt the membrane with the pipette tip and pipette only from the edge of filter.
I would advise to use commercial filters that have a so-called dead space volume. This means it is not possible to dry out the sample completely. I personally used the commercial Sartorius™ Vivaspin™ centrifugal filters since they have built-in dead stop pockets.
Troubleshooting Your Centrifugal Filters
Precipitation of Protein
One of the common problems that people face when using centrifugal filters is the issue of precipitation of protein. This primarily arises because of the formation of a gradient of protein while concentrating and a local high concentration near membrane. This can be avoided by mixing the solution at regular intervals as mentioned above.
Also, make sure that the pH of the buffer is not too close to the pI (isoelectric point) of your protein of interest.
Finally, ensure that the DTT in the buffer is freshly made. DTT is unstable at pH 8.0 and over time may become inactivate. Consequently, formation of disulphide bonds may start in random and unnatural ways and hence forming different disulphide oligomers of proteins, which may be insoluble.
Loss of Protein
Loss of protein is another one of the common problems that is encountered after concentrating a protein using centrifugal filters. The first obvious mistake could be that the starting amount of protein is too low. If you want to use filters when you have a very low amount of protein (e.g. 1-10 µg), it is crucial to go through a process called surface passivation. This involves pre-treating the centrifugal filter overnight with 5% Tween® 20 (v/v) in ultra-pure (e.g. Milli-Q®) water and then soaking/rinsing with ultra-pure water. The unit can then be used immediately or stored further in PBS (top and bottom filled) for months. Many people also suggest using 2% BSA instead of Tween 20 for this purpose.
Binding of the Protein to the Centrifugal Filter Membrane
This property is largely dependent on the composition of the membrane of the filter, whether it is polysulfone, polyestersulfone or regenerated cellulose. Personally avoid regenerated cellulose as I found binding of the protein to the membrane more of a problem with these filters. Many people add 0.1% (v/v) Tween 20 or Triton X-100 to mitigate this problem. It is tempting to use several centrifugal filters at the same time in order to increase the speed of concentrating your sample, but it is not a good idea since this can increase loss due to the potential problem of binding of protein to membrane.
Finally, after concentrating the protein, check the concentration either by taking the absorbance at 280 nm or using a Bradford assay, whichever is convenient and available.
I hope these handy tips and tricks related to centrifugal filters helped you to troubleshoot your experiments. If you have any tips for using centrifugal filters, please leave a comment below.
Image Credit: Luke Hayfield