Clontech Laboratories His60 Ni Superflow Cartridges Podręcznik użytkownika - Strona 5

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His60 Ni Superflow Cartridges User Manual

Table 2. Reagent Compatibility with His60 Ni Superflow Resin (Based on Literature References)

Reagent
Notes
Amino Acids
Arginine, Glycine,
Glutamine
Histidine
Binds to His60 Ni and competes with histidine residues in the
his-tag.
Buffers
HEPES, MOPS
Amine groups that are present in these buffers can interact with
Ni
Sodium acetate
Sodium phosphate
Tris
Coordinates weakly with metal ions, causing a decrease in
binding capacity.
Chelating Agents
EDTA, EGTA
Will strip metal ions from the resin, resulting in protein elution
and a resin color change.
Denaturing Agents
Gu-HCl
With high concentrations, protein unfolding generally takes
place. Protein refolding on-column (or after elution) is protein-
dependent.
Urea
With high concentrations, protein unfolding generally takes
place. Protein refolding on-column (or after elution) is protein-
dependent
1
Detergents
CHAPS
Ionic detergents such as CHAPS, SDS, and sarkosyl are
compatible at concentrations up to 1%. Even at low
concentrations you should expect interference with binding.
NP-40
Has high absorbance at 280 nm.
SDS
Ionic detergents such as CHAPS, SDS, and sarkosyl are
compatible at concentrations up to 1%. Even at low
concentrations you should expect interference with binding.
Triton X-100
Has high absorbance at 280 nm.
Tween 20
Reducing Agents
ßME
Use the resin immediately after equilibrating with buffers
containing βME. Otherwise the resin will change color. Do not
store the resin in buffers containing βME. A slight change in
color (yellowing of the resin) will occur.
DTT
Since DTT is a reducing agent, low concentrations will reduce
the metal ions in His60 Ni Superflow resin. Although enough of
these ions may remain unaffected to allow protein purification,
please use it with caution. Do at least 20 column volumes of
washes, preferably with low concentrations of imidazole (40
mM) to wash out any reduced metal ions.
DTE
Others
MgCl
2
CaCl
2
Ethanol
May precipitate proteins, causing low yields & column clogging.
Glycerol
1
Detergents cannot be easily removed by buffer exchange.
PT5143-1
031716
2+
ions, diminishing the resin's binding capacity.
Clontech Laboratories, Inc. A Takara Bio Company
www.clontech.com
Acceptable Concentrations
Not recommended
Can be used at low concentrations (20
mM) to inhibit nonspecific binding;
and, at a higher concentration (up to
100 mM), to elute his-tagged proteins
Up to 100 mM (with caution)
Up to 100 mM (with caution)
Up to 50 mM can be used
Up to 50 mM (with caution). Loss in
binding capacity can be seen.
Not recommended
6 M
8M
1% (with caution)
2%
1% (with caution)
1%
2%
20 mM (with caution)
1 mM (with caution)
Not recommended
4 M
5 mM
20%
20%
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