Summary

• PhyTip gel filtration columns operate as small scale, chromatographic separation columns.
• They are the only high throughput, automatable, chromatography desalting solution.
• Similar to the PhyTip affinity columns, the resin of choice is positioned between two inert screens that maintain the resin bed at the base of the column.
• Unlike the affinity columns, the sample is passed unidirectionally through the column by gravity flow.

Product Benefits

High Performance:

• Remove >95% of salts with >80% yield of protein
• Retain protein functionality
• <10% CV for volume and concentration between final samples
• Ready for downstream assay

Automated desalting/buffer exchange of functional proteins:

• Fully automated process with MEA Personal Purification System
• 12 samples desalted/buffer exchanged in ~30 minutes.

High throughput format:

• Available in 96 sample format for high throughput processing

Sample Separation

Figure 1. Sample (top tube #1) containing brown myoglobin protein (16.7kDa) and yellow DNP-glutamate salt (313Da) was loaded onto a 600uL PhyTip gel filtration column. The same PhyTip column at different steps of desalting is pictured along with the collected flow through. From left: 1) column conditioned by PBS buffer prior to sample loading, 2) column after 200μL sample has entered the resin bed, 3-6) column after 100μL PBS buffer is applied, 7) column after a final chase of 400uL PBS buffer. Microfuge tubes from left: 1) 200μL starting sample, 2) flow through collected after sample is applied, 3-6) fractions collected after each 100μL chaser, 7) fraction collected after final 400μL chaser, to remove all protein. This example illustrates a typical separation allowing the user to discard sample flow through (2) to maintain concentrated purifications. The salt is retained until fraction (6), while most of the protein is released by fraction (4).

The process:

1. Column Equilibration: PhyTip gel filtration columns are packaged in a shipping fluid designed to maintain proper hydration of the size exclusion resin. The shipping fluid is replaced with the buffer or solvent of choice by equilibrating the column by soaking the box in the solvent overnight.
2. Sample Loading: The sample is applied to the top of the column using a transfer pipette tip. The sample enters the column by gravity.
3. Elute: A chaser volume is added to the top of the column following sample loading to allow the sample to fully enter and exit the column. Discrete chromatographic fractions can be collected as they are eluted from the column.