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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

1 2 ... 158 159 160 161 162 163 164 165 166 167 168 ... 299 300

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Bio-rad ChromLab™ Software Manual de usuario Pagina 163