Spinning the centrifugal filters: Tips and Tricks
One of the most widespread protein laboratory accessory is the MWCO (molecular weight cut-off) centrifugal filters which is commonly used for concentrating protein and DNA too. They are available commercially with different cut-offs like 3kDa, 30kDa, 50kDa, 100kDa and so on. These little devices are expensive and hence demands proper usage and care to utilise its benefit to full extent. Here, I am going to discuss some tips and trick pertaining to its storage, use, and finally some troubleshooting inputs gathered from my experiences with centrifugal filters.
1) Storage: One of the most important question that comes in mind is to how to store the centrifugal filter after use? Remember, if you show a little care you can probably save few bucks of your supervisor and probably much of your precious experiments. 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.
My personal experience of adding PBS (1X) with little sodium azide (0.02% w/v) was most helpful for me. Apart from this, you can also add 20% ethanol and then store it. Nevertheless, it is crucial to store the filter at 4 degrees (not in freezer or -20 degrees). The polymer structure of those fragile polymers used to form the membrane are prone to damage by repeated freeze/thawing.
2) Cut-off: As said earlier in the article, the centrifugal filters are available commercially in different cut-offs. It is important to realize that MWCO filters are not meant for separating different molecular weight entities. For separating molecules on basis of molecular weight, we have well established 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 your desired molecular weight species which I want to concentrate.
However, if you use very low cut-off, it will take long time and subsequent refilling. Moreover, if you use something like 3000 Da (3 kDa) cutoff, there is possibility of concentrating low molecular weight solutes as well as proteins, sometimes potentially contributing to protein precipitation. On the contrary, if you use high cut-off, there will be more membrane surface which can provide more area for non-specific binding.
3) Reuse: Owing to the price constraints, it is impelling to use used MWCO filters again. Although not ideal, it can be reused if they don’t clog. In spite of this fact, it should be discarded after maximum 3rd use. These filters even start to leak after use of 2-3 times. After use, it is imperative that proper storage conditions discussed above are followed.
4) Use: When starting to concentrate large volumes, I personally give spin for 5 minutes to determine how fast the solution is flowing through the filter and then decide time to give the spin. It is better to give a spin for 15 minutes initially and then with pipette gently mix the sample 2-3 times to avoid blocking of membrane. It is better not to lose your hard work and money at this stage. You will see 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 pipette tip and pipette only from edge of filter.
I advise to use commercial filters that have a so called dead space volume, by which it is not possible to dry out the sample completely.
5) Precipitation of protein: One of the common problem that people face is the issue of precipitation of protein. Mostly this case arises because of gradient of protein forming while concentrating and local high concentration near membrane which can be avoided by mixing the solution at regular interval. Also, make sure that pH of the buffer is not close to pI (Isoelectric point) of protein of interest. Apart from this, make sure that DTT in the buffer is freshly made. DTT is unstable at pH 8.0 and over time may get inactivated. Consequently, formation of disulphide bond may start in random and unnatural way and hence forming different disulphide oligomers of proteins which may be insoluble.
6) Loss of protein. It is also one of the common problems that is encountered after concentrating the protein. The first obvious mistake could be that starting amount of protein is very less. If you want to use filters in such a case, it is crucial to go through a process called surface passivation. It involves pre-treating the unit overnight with 5% Tween20 (v/v) in mQ water and then soaking/rinsing with mQ water. The unit can then be used immediately or stored further. Many people also suggest using 2% BSA instead of Tween20 for this purpose.
7) Binding of protein to membrane: This property is largely dependent on membrane composition of filter; whether it is polysulfone or polyestersulfone or regenerated cellulose. Different chemistry may be ‘kinder’ of your proteins. I personally suggest not to go for regenerated cellulose as I found this problem to be graver there. Many people add 0.1% (v/v) Tween 20 or TritonX-100 to mitigate this problem. It is tempting to use many centrifugal filter unit at same time to increase the speed of concentrating but it is not a good idea since there can be more loss due to the potential problem of binding of protein to membrane.
Finally, after concentrating the protein, go for absorbance at 280 nm or Bradford assay whichever is convenient and available.