Clontech Laboratories His60 Ni Superflow Cartridges Podręcznik użytkownika - Strona 15

Przeglądaj online lub pobierz pdf Podręcznik użytkownika dla Sprzęt laboratoryjny Clontech Laboratories His60 Ni Superflow Cartridges. Clontech Laboratories His60 Ni Superflow Cartridges 15 stron. Purification of expressed his-tagged proteins from bacterial, yeast, mammalian, and baculovirus-infected cells.

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His60 Ni Superflow Cartridges User Manual
VI.

Troubleshooting Guide

Table 3. Troubleshooting Guide for His60 Ni Superflow Cartridges

Description of Problem
Low target yield
Impurities in eluate
Low flow rate
Can not detect target protein by
UV
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PT5143-1
031716
Possible Explanation
Poor expression of target protein
Target protein forms inclusion bodies
Inefficient target extraction
Inaccessible polyhistidine tag
Insufficient washing
Clogged cartridge
Viscous sample
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Solution
Optimize bacterial expression
conditions.
Decrease temperature to 25°C or
lower during induction to
minimize inclusion body
formation.
Solubilize inclusion bodies and
perform the purification in the
presence of 8 M urea or 6 M
guanidinium HCl.
Use our xTractor Buffer.
Purify in presence of 6–8 M urea or
6 M guanidinium HCl.
Increase wash volume or add
intermediate wash at 60 mM
imidazole. (This can result in partial
loss of target protein.)
Apply only clarified extract, and
decrease the amount of loaded
sample.
Treat sample with Benzonase or
DNase I, as described in Section V.A.
Use low UV absorbance
imidazole in the buffers.
Perform a Bradford protein assay
on collected fractions to identify
target protein in eluate.
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