Lidstrom:His-tag Protein Purification

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Cobalt versus Nickel Resin

• Ni has better binding capacity (~3X) but Co has better specificity (higher purify product)
• Use Co unless you have a compelling reason to use Ni

Increasing binding capacity

• Adding sodium chloride or glycerol can help increase your protein yield by reducing the extent to which other proteins can stick to the resin.
• This experiment shows compares purification of His-tagged ACS in a 50mM pH 7.5 phosphate buffer with either 0mM NaCl, 100mM NaCl, or 500mM NaCl added. The NaCl was added to the lysis buffer, and the first two of the three wash buffers (all 10mM imidazole, but varying [NaCl]).
  • The yield is much higher for 500mM:

    

    Higher [NaCl] can give higher yields of the desired protein. The y-axis shows the total protein eluted (uL of elution * uM of elution), which sums the elutions that were kept separate.

  • The gel of the flow-through (what didn't stick to the resin) shows that more ACS bound in the 500mM NaCl solution. [[image:140516_SDS-PAGE_vary_NaCl.png|thumb|upright=2.0|center|Vary [NaCl] when purifying ACS.
  • The data and analyses for this are found here and here but I haven't tidied it up for others to view.