Bio-Rad Profinity IMAC Resins - Manual (Page 22)

4. Wash the cartridge with 6 CV of wash buffer 2
at 2 ml/min.
5. Elute the purified protein with 10 CV of elution
buffer at 2 ml/min.
6. Prior to quantitation of the protein concentration,
the purified protein should be exchanged into a
non-imidazole buffer (imidazole can absorb at
280 nm). Purified protein from denaturing
purifications should be exchanged into another
buffer through dialysis.
The chromatogram and gel in Figure 5 illustrate a
representative purification of a high-expressing
soluble protein purified using the native buffer set
and method described in Table 6. Note: IMAC
buffers made with potassium salts are more stable
than sodium salt-based buffers. However, potassium
will complex with SDS in Laemmli buffer and
precipitate out of solution. Prior to analyzing IMAC
samples on gels, the samples must be diluted at
least 1:7 with Laemmli buffer to prevent precipitation.
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