1. Affinity purification of Poly-His tagged proteins

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Salt concentration changes with respect to end goal of the purification. Use a midpoint to start and then go higher or lower on a case-to-case basis.

 

Lysis buffers: Buffer A (50 mM Tris–HCl (pH 7.5), 150 - 500mM NaCl, 30 mM Imidazole and 1 mM DTT) + 0.5 mg/ml Lysozyme, 40 U RNase free DNase, 1 mM phenylmethanesulfonyl fluoride or other Protease inhibitor cocktails.

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Wash buffer: Buffer B (Buffer A (50 mM Tris–HCl (pH 7.5), 150 -500 mM NaCl, 80 mM Imidazole and 1 mM DTT) .

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Elution buffer for linear concentration gradient: Buffer C (Buffer A with 250 to 500 mM Imidazole). Use it with buffer A to make gradients on a chromatography system.

proteins

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A rough outline for a prep :

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Day 1:

-Harvest and freeze cells after overexpression at desired conditions. 

-Resuspend frozen cells in Buffer A.

-Lyse by sonication or Cryo pulverization also works very nicely. Lately that has become my favorite. 

-Spin down lysate at max speed (about 30,000g) for 30min at 4 degree C. Use Oakridge kind tubes

-Collect supernatant by pouring it into fresh Falcon tube

-Load on a HisTrap column or beads for suggested time periods. 

-Wash with Buffer B

-Elute directly or with a linear imidazole gradient.

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2. Affinity purification of Strep-tagged proteins

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-The tag can give very god purity levels but tends to be costlier alternative than His. One could sometimes skip intermediate chromatography steps and proceed to polishing after Strep pulldowns.

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-Buffers and lysate preparation can be similar to the Histag method above, with an exception of not using imidazole in any buffer and eluting with Desthiobiotin.

 

- Note bacterial lysates tend to have some small amount of biotin and may compromise the column over a period of time by irreversibly binding to the streptavidin trap on the column.

-Be precise with column regeneration parameters as it uses NaOH and the column has an active protein that traps the strep tag. 

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3. Other Affinity purification alternatives

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- Constructs with solubility tags can also be used for affinity pulldowns. Eg . MBP/SUMO can be used to purify proteins.

- Similarly, GFP/mCherry fusion proteins can also be used for pulldowns with specific nanobodies immobilised on sepharose beads.

- Among epitope tags, I have only used 3xFLAG and its works beautifully. It is much costlier though.

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