Software Manual

Standard Method Templates
User Guide | 161
Chromatofocusing Chromatofocusing chromatography uses a charged matrix to
bind protein molecules. A pH gradient is used to elute the
bound proteins, which elute when the pH is the same as the
pI of the protein of interest (overall charge = 0). Special buffer
systems are required to perform the pH gradient over a large
range.
Desalting Desalting is usually used for buffer exchange. Proteins do not
bind to the column matrix and ar
e typically eluted isocratically
in the void volume of the column. Select a buffer system that
maximizes the stability of the target protein.
Hydrophobic Interaction Hydrophobic interaction chromatography uses high salt
buf
fers to adsorb target proteins to a hydrophobic column
matrix. Decreasing salt concentrations are then used to elute
and separate bound proteins.
Mixed Mode Mixed mode chromatography uses a column matrix with
hydr
ophobic and charged ionic interactions. Proteins can be
eluted using a gradient of pH (eluting when the pH = pI of the
target protein) or salt (increasing salt to elute from the
charged moiety or decreasing salt to elute from the
hydrophobic moiety of the column matrix).
Multicolumn Sequential Multicolumn sequential purification uses these templates
when
multiple samples must be purified on multiple
columns. The samples are injected sequentially either by
using a sample pump with sample inlet valve or through
sample loops. Each sample is loaded onto a column and
washed to remove contaminants that can cause sample
degradation. The columns are then eluted using either
step or linear gradient protocols in a sequence. The
fractions are collected with the BioFrac™ fraction
collector or an outlet valve.
Table 6. Standard Method Templates, continued
Method Template Explanation