Home-made Ni2+ columns for His-tagged protein purification

UPDATE 10/29/14: The Cube Biotech columns apparently break VERY easily when taking the end pieces in and out, especially on the second or third round of packing them. I would not recommend them anymore. We are trying out the Econo-columns from Bio-Rad, which cost about the same but are only good for use in low-pressure environments.

We purify a lot of proteins, and the vast majority are cloned with His-tags for a simple affinity purification using immobilized metal-affinity chromatography (IMAC), usually with a Ni2+ resin. Since starting the lab, we have found the best combination of speed and ease-of-use with 5 mL pre-packed columns, which we use with a peristaltic pump. You can do several preps at a time, and using the pump gives you reproducible flow rates and prep times. However, these columns are pretty expensive: around $540 for 5 x 5 mL columns from either Qiagen or GE, making each one around $107. This adds up quick.

Recently I found these empty 5 mL columns at Cube Biotech:

Cube Biotech PureCube empty cartridge (#16917)

Cube Biotech PureCube empty cartridge (#16917)

These are $174 for five, or $34.80 each. With the 5 mL bed filled with Ni-NTA agarose from Qiagen, the total price of a home-made column is $76.40 (we paid $832 for 100 mL Ni-NTA agarose, making 5 mL cost $41.60). Filled instead with Ni-IDA resin from Machery-Nagel (which works really well; at least as pure after the Ni2+ step as with Qiagen or GE resin), the total price is $50.35 (we paid $187 for 30 grams of Ni-IDA resin; 2.5 grams needed to fill this column costs $15.55).

$50.35 on the left, $107 on the right. Can you tell the difference?

$50.35 on the left, $107 on the right. Can you tell the difference?

An added benefit - the columns are re-usable, so when your resin reaches the end of its useful life, you can refill the column and bring your cost down even further.

Happy prepping!

Here we are!


Over the past two (almost three) years since I started my lab, a lot has happened. Our bread and butter is crystallography, and we've collected just about 1.2 Terabytes of diffraction data in over a dozen trips to the Advanced Light Source, the Stanford Synchrotron Light Source, and the Advanced Photon Source (thanks, Department of Energy!):

We'll have a few things to say soon, stories that we think are really interesting and we are excited to share. While these stories wind their way through the academic-publishing maze, I plan to use this space to share my experiences starting up a new crystallography & biochemistry lab at UCSD. This will include setting up a web site, managing shared documents in a small lab, and storing and backing up all our diffraction data in a way that doesn't keep me up at night (too much). If you're curious about our work in the meantime, please visit our structure gallery, publications page, or lab web site at Ludwig Cancer Research.