Bio-Rad Profinity IMAC Resins - Manual (Page 24)

Fig. 5. Native IMAC purification: A 32 kD Nif-3* His-tagged protein was
purified from the soluble fraction using the standard Profinity IMAC native
purification protocol. 2 ml of lysate (2 CV) from a 100 ml E. coli culture was
loaded onto a 1 ml IMAC cartridge. The cartridge was washed with 6 CV of
wash buffer 1, followed by 6 CV of wash buffer 2, and purified protein was
eluted with 5 CV of elution buffer (all at 2 ml/min). The purified product was
>95% pure by densitometric scanning and Quantiy One
® software analysis.
Lane 1, Precision Plus Protein unstained standards; lane 2, soluble lysate;
lane 3, Flow-through; lane 4, wash 1; lane 5, wash 2; lane 6, purified product.
*Nif-3 construct was kindly provided by R. Stevens, Scripps Institute.
Section 9
Scaling Up
Bio-Scale Mini cartridges are available in 1 ml and
5 ml cartridge formats. The Profinity IMAC resin is
also available in larger amounts, from 25 ml bottles
to bulk quantities, for scaling up methods developed
using the cartridges.
For quick scale-up, two or three cartridges of the
same type can be connected in series; backpressure
will increase with cartridges in series, so care
should be taken to maintain an overall system
pressure
≤45 psi.
In addition, Bio-Rad carries an extensive line of
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