Roche cOmplete His-Tag Purification Resin Column Reference guide

Brand: Roche

Model: cOmplete His-Tag Purification Resin Column

Type: Reference guide

For life science research only. Not for use in diagnostic procedures.

COMPLETE is a trademark of Roche.

All other brands or product names are trademarks of their respective holders.

Determine the optimal operating conditions for the specific target protein.

Both purity and yield depend on the sample amount. If the amount of sample is too high, the resin’s binding capacity

may not be sufficient to bind all target protein, resulting in a suboptimal protein yield. If the amount of sample is too

low, the remaining binding sites on the resin may enable background binding of lysate components.

Optimal results are obtained when the amount of target protein matches the amount of resin

(in columns or as loose resin).

Determine the optimal ratio of lysate and resin volume within the column or resin

volume for loose resin.

The capacity for a given target protein depends on several factors such as

 protein size

 conformation and multimerization status

 length and accessibility of the his-tag

For best results when purifying a specific protein of interest, refer to the following table:

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cOmplete His-Tag Purification Products

Quick Protocol

For products with catalog numbers:

06781535001 1 column (5 ml resin) 06781543001 5 columns (1 ml resin each)

05893682001 25 ml settled resin volume 05893801001 200 ml settled resin volume

Pre-Packed Columns Resin

Incubate the columns with varying volumes of lysates, in parallel

test experiments.

Incubate a small quantity of settled resin with varying

volumes of lysates, in parallel test experiments.

Wash the columns and elute the bound proteins. Wash the resin and elute the bound proteins.

Determine the amount of target protein in the unbound fractions

and in the eluate using SDS-PAGE.

Determine the amount of target protein in the unbound fractions

and in the eluate using SDS-PAGE.

Result Interpretation

The volume of lysate is optimal when only a small amount of target

protein remains in the flow through, and the maximal amount of

protein is detected in the eluate fractions.

The volume of lysate/volume of resin ratio is optimal when only a

small amount of target protein remains in the flow through, and the

maximal amount of protein is detected in the eluate fractions.

The parameters allowing for the maximal protein yield and purity vary significantly depending on the protein’s

characteristics. For best results, optimize the key parameters in small-scale trial purifications.

Additional tips

 Start without imidazole in loading and washing buffers. If improvement of purity is needed, use imidazole up to

5 mM in a second step.

 The yield of the target protein can be optimized by allowing more time for the protein to bind to the resin.

This can be performed by reducing the flow rate during the loading step.

 Optimal results can typically be achieved with buffers containing a high-salt

concentration (300 mM NaCl) at pH 8.0 for target proteins compatible with

those conditions.

Note: The pre-packed cOmplete His-Tag Purification Columns are fully

compatible with standard FPLC systems (e.g. ÄKTA Systems, GE Healthcare).

 expression level and solubility of the his-tagged protein

 lysate concentration

 buffer pH and composition

Roche cOmplete His-Tag Purification Resin Column Reference guide

Roche cOmplete His-Tag Purification Resin Column Reference guide

Roche cOmplete His-Tag Purification Resin Column Reference guide

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