Editing Protein Purification 3 Steps (section)

=== <br>Polishing ===

<br>Gel filtration (GF)

HiLoad 16/60 &amp; 26/60<br> <br> <br>Settings that improve purification by Size exclusion chromatography

Having the best column for your needs doesn’t necessarily mean that your purification step will be ideal with ~100% resolution (the measure of separating one specie from another). In many cases some contaminates will be evident, some even in a cryptic shoulder that will be very difficult to observe. Here are the main settings you should give attention before running a separation:

• Load – avoid overloading the column with your precious protein unless you know there will be no close species to your protein’s elution volume. Seek advise with the column’s data sheet in regard to acceptable injection volume and protein mass. Measure the sample’s concentration and if the sample is not pure add 10-20% mass to your final protein concentration (according to the amount of contamination. As a rule of thumb your protein sample volume and mass for injection should be in the range of 0.5-3% of the column volume and no more than one tenth of column volume in terms of mass. This means that if you’re using a 24ml column, use 120-500 microliters of protein sample with a maximum protein mass of 2.4mg. Again, these estimates are for your first run and if you find you can inject more protein mass, then go ahead. However, if you find a cryptic shoulder (a sign of a closely eluting specie) then you might want to lower the amount of protein you inject. It will be easier for you to cut out a small peak of several injections than trail than a larger trail in a single overloaded injection.

• Flow rate – chromatography is based on the dynamic transition between equilibrium and disequilibrium between the mobile and the stationary phases (“theoretical plates”). For this equilibrium to occur, sufficient time should be supplied. With respect to our modern FPLC systems, this means a reasonable flow rate, which depends on the specific column (usually no lower than 0.2ml/min or higher than 4ml/min).

• Mid-run injection – In cases you have to do many injections you might want to explore mid-run injection. As the name implies, after performing the first separation analyze whether you can inject an additional sample before or after the expected elution volume. For example, if you have an elution profile of peak A eluting at 7ml with peak width of 1ml, peak B starting eluting at 12ml with a peak width of 1ml and peak C eluting at 20ml with peak width of 0.5ml, you can safely inject two injections 2ml apart. This technique can save a lot of time when the chromatogram is not crowded with many peaks.

• Consecutives SEC columns – In case you have a combination of complexes (80kDa) and monomers (40kDa) you might want to explore the use of two complementary columns, such as Superdex 75 and Superdex 200. Assuming you’re aiming at isolating the complex from the monomers and the other small contaminants, the first separation should be performed with Superdex 75, collecting the complexes eluting at void volume and then reinject the elution into Supderdex 200 to separate them from the other contaminates. This technique is especially effective when it is hard to separate complexes from monomers in a single column.

Convert linear flow units (cm/hr) to volumetric flow units(ml/min). <br>Formula for the conversion from cm/hr to ml/min (VF):<br>VF=0.013*X*d^2 where X is the linear flow value and d is the internal diameter of the column in centimeters.<br><br>

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