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

General Info

Acceptable pH range for buffers

• Histidine pKa is ~6. At pH8, most of the His residues are deprotonated and stick more strongly to the Co Resin. This also means that undesired proteins with His are more likely to stick at higher pHs.

Other optional additives

• NaCl
• glycerol
• DTT

Don't ever add

• TCEP, DTT, or other strong reducing agents. It will reduce the resin and it will turn a funny color.
• Chealating compounds.
  • EDTA (a potent inhibitor of metalloproteases) can only be added in tiny amounts. Be careful about Tris, ammonium salts, and certain amino acids.

What is a "good yield"?

• Justin Siegel says 10 g/L is good. But this depends on how much buffer you eluted in. If you dilute in twice as much volume, your concentration might be 1/2 as high.

Increasing binding capacity

Sodium Chloride

• 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. This data is from flask-scale purifications.

  • The gel of the flow-through (what didn't stick to the resin) shows that more ACS bound in the 500mM NaCl solution.

    

    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.
  • However, the same findings did not hold true for my high throughput purifications of the same proteins.

    • 

      Vary [NaCl] when purifying ACS with L641P mutation. Data & analysis

Glycerol

Glycerol can increase your yield via similar principles.

Experimental tips

• 2M imidazole can be made and stored in the fridge for weeks/months to prepare the wash and elution buffers. Reserve about 10% of the volume for adjusting pH down with full strength HCl. For instance, ~10-15mL of HCl is needed to make 100mL of 2M imidazole, pH 7.4 in water.
• Adding NaCl lowers the pH of a solution. It