Manual purification of proteins using anion exchange chromatography
The minimal equipment needed is an empty open-top glass chromatography column with a support frit at the bottom and a stopcock (Bio-Rad Econo-columns are inexpensive and come in a range of sizes). A ring stand and clamp can be used to support the column in a vertical orientation. Select a resin, prepare it according to the manufacturer's instructions, and pour it carefully into the tube, avoiding bubbles. Let the resin settle under gravity while draining. Don't let the column run dry.
An inexpensive resin is Whatman DE52 DEAE-cellulose. Be careful when handling it not to mix it around too vigorously because it will break up into small particles (fines) that will slow down the column elution rate. Prepare it by gently suspending it in the starting buffer, then letting most of it settle for a short while, Pour off the unsettled particles and repeat a few times. This is to remove the fines. Use a glass rod or spatula to resuspend the resin. Never use a magnetic stirrer, which will break it up. QAE-Sepharose is another good, relatively inexpensive anion exchange resin.
At the start, the ionic strength/salt concentration should be low (no salt). Apply the protein sample to the top of the column and allow it to drain in slowly. Avoid disturbing the top of the resin when applying the sample. Collect fractions in polypropylene tubes, such as microfuge tubes. Wash the column with more buffer by adding it to the top once the sample has all drained in. Elute the column with buffer containing gradually increasing salt concentrations. You can do this using steps of salt concentration if you have no way to form a gradient, but it is better to use a linear gradient (see below). If you use steps, add a small amount of buffer containing each salt concentration and let it drain in. Then add the next higher salt concentration. Keep the steps small to simulate as closely as possible a linear gradient. (e.g. 10 mM, 20 mM , 30 mM, etc).
Two-chamber gradient formers of various sizes can be purchased, but you can make one with 2 beakers, a magnetic stirrer, and a bit of Tygon tubing. One beaker containing the low-salt buffer is magnetically stirred and is connected by tubing to the top of the column, which is sealed with an airtight cap. The other beaker contains the high-salt buffer and is connected by tubing to the low-salt beaker. The connecting tubing must be filled with one of the buffers at the start because it is going to be a siphon. The beakers should be at the same level and above the level of the column outlet. There should be only a minimal amount of liquid at the top of the column so that as drops fall in the salt is not diluted.
Once the column is eluted, you can measure the 280 nm absorbance of the fractions one at a time with a spectrophotometer to obtain the chromatogram. SDS-PAGE can be used to see where your protein eluted, if is abundant enough. If your protein is an enzyme, you may be able to do an enzyme assay on the fractions to find out which ones have your protein. If it is a low-abundance protein and you don't have an assay for it but you have an antibody against it, you can follow it by doing ELISA or Western blots.
@ Adam,
Thanks for your support. Dear Adam, what is the best media for manual anion exchanger? how it is decided? and how to decide the retention period and elute the protein?
Hi Bhagya,
I suggest you read this handbook about the technique for more details.
http://www.gelifesciences.com/gehcls_images/GELS/Related%20Content/Files/1314823637792/litdoc11000421_20140416231512.pdf
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