Lidstrom:His-tag Protein Purification: Difference between revisions
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(New page: Back to Protocols == Cobalt versus Nickel Resin == == Increasing binding capacity == * Adding sodium chloride or glycerol can help increase your protein yield by ...) |
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Back to [[Lidstrom:Protocols|Protocols]] | Back to [[Lidstrom:Protocols|Protocols]] | ||
== Cobalt versus Nickel Resin == | == 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 == | == Increasing binding capacity == | ||
Revision as of 09:14, 16 May 2014
Back to Protocols
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:
Vary [NaCl] when purifying ACS. Shows much higher ACS binding at 500mM NaCl - The gel of the flow-through (what didn't stick to the resin) shows that more ACS bound in the 500mM NaCl solution.
- The yield is much higher for 500mM:
