Protein Purification and Method Development Using PhyTip Columns
Protein Purification and Method Development Using PhyTip Columns

Getting Started : Sample and Column Preparation and Use

Why are PhyTip columns shipped with some fluid in them? What is the fluid?

PhyTip columns are delivered with glycerol shipping fluid in order to keep the resin bed hydrated which is necessary for optimal performance. The PhyTip columns may be used straight out of the box without preconditioning. However, the first step of a programmed method is usually an equilibration step to ensure the capture step that follows with under identical conditions column to column. The equilibration step is usually performed with a buffer that is similar to the sample being processed.

Is it necessary to condition the PhyTip columns before the capture step?

It is not mandatory but recommended in most cases. The equilibration step dilutes the glycerol that is used to pack the resin. The pH of the resin/solution is optimal for the subsequent steps.

How clear or clean should my sample be before I begin to use a PhyTip column?

PhyTip columns have been used with a variety of different sample media. In all cases, we recommend that the solution to be purified is clear of any particulate substances. In order to guarantee this, we recommend the sample is centrifuged and then the clear supernatant processed by the tip. Care should be taken not to disturb the plug of spin down residue while sample is processed. After centrifugation, it is recommended to transfer the sample to a fresh well before processing.

What is the maximum sample volume I can process with my PhyTip column?

For the 1000+ tips we recommend a maximum of 1 mL sample and for the 200+ tips a maximum of 200 μL sample. Larger samples can be processed by aliquoting sample into multiple wells of a plate which are then processed sequentially. For example, a researcher purifying 5 mL of sample is recommended to perform 5 separate capture steps. First, aliquot the protein sample into five separate 1 mL volumes. Then program the robot to perform a capture step on each of these individual aliquots. In this way, there is a maximal capture of all of the protein in the original 5 mL. The subsequent wash and elution steps can proceed as normal. This general process may be adapted to different sample volumes as desired.

How do I know if my resin is capturing protein? How do I know if my resin is saturated with protein?

If used correctly, PhyTip columns can be loaded with a surprising amount of protein.
Nevertheless, in some cases the resin may have the incorrect selectivity and no protein may be captured. In other cases, protein is captured well loading up the resin, but not all of the protein is collected from the sample volume. This is difficult to observe directly as most proteins are colorless and only after the fact can capture information be determined. Because of parallel operation of PhyTip column, the user is in complete control. Protein control standards may be run in parallel with the sample to ensure correct operation and purification chemistry. In a broader experiment researchers will retain some of the original material, the “flow-through” from each step (this is the material left after a step) so that a gel can be run at the end of the experiment to observe not only the end product – the purified, concentrated protein, but also if any of the protein of interest came through in the flow through. Reviewing this information allows the researcher to determine the success of the capture, wash and ultimately the elution steps.

How do I program my robotic system? For example, can I elute with an elution volume that is less than 3 x of the resin column bed volume? 

Various default method protocols are provided by PhyNexus and excellent results can be obtained with no method modifications. However, it is possible to make programming adjustments or even major adjustments to make a new method. For example, it may be desired to increase the concentration of the recovered protein by with only 2 x the resin volume rather than the normal 3 x volume. Or larger or multiple elutions may be desired to ensure complete recovery of the protein. If programming is method development is not available within company resources, see your PhyNexus representative for advice and help with robotic programming.

Why is flow rate important when operating the PhyTip columns?

A critical component of the capture step requires that the target sample protein have sufficient time to bind to the affinity resin. By using a recommended slower flow rate, maximum capture of the desired protein can occur because this increases residence time of the sample protein with the resin matrix. The default protocol utilizes a residence time of 6 and 18 minutes for 200
μL and 1000 μL samples, respectively. The capture efficiency can also be increased by increasing the number of capture cycles.