Hello,
I am pretty new to protein purification and would appreciate some clarification on anion exchange chromatography using GE DEAE Sephacel.
1. Is gravity flow sufficient for this experiment?
2. How much of DEAE sephacel would I have to put on my column?
3. My protein's isoelectric point is 5.07. The buffer I have chosen is piperazine with pkA 5.7 at 25 degrees C. I am planning to adjust pH to 6.07 for this experiment. Does this seem right?
4. How do I determine best flow rate?
5. What NaCl concentrations is best to elute my sample ? The recommended is 0-.5M, but I was wondering how to approach choosing the right concentrations.
Thanks so much for your time!
As usual,
an undergraduate researcher with many questions
Have a great day!
The manufacturer's instruction manual for the resin is here:
https://www.gelifesciences.co.jp/tech_support/manual/pdf/71710000.pdf
The size of column required depends in how much protein is going to be applied to the column. The column should be filled with resin nearly up to the top, leaving room for putting the top on. Ideally, there would be no headspace. A long column is not necessary for high resolution in ion exchange chromatography, unlike gel filtration chromatography.
The flow rate should be chosen not to exceed the maximal linear flow rate.
The salt gradient for elution is determined experimentally. People often start with a gradient up to 1 M NaCl, but if the protein elutes at a low salt concentration, a shallower gradient may be used to improve purity.
Personally, I would use a pH closer to 7 if the protein is normally present at neutral pH, because it will probably be more stable. That will require using a different buffer, such as HEPES or MOPS.
The chromatography should be performed in the cold, if possible, to reduce protein denaturation and proteolysis.
The ionic strength of the extract is an important factor when binding protein to the column. If the ionic strength is too high, the protein may not bind. Do not put 150 mM NaCl in the lysis buffer, for example.
Sorry, typo. pI of protein is 5.07.
Thanks Dr. Shapiro! This is very helpful.
Couple of comments.
Gravity flow is fine if you can live with large processing time. Piperazine will not buffer at your selected pH. Do not use mops or hepes as they will compete for binding. Run at cold conditions only if you know that you protein is degrading fast.
"Recommended Buffers for Anion Exchange Chromatography"
http://wolfson.huji.ac.il/purification/buffers/Buffers_AEIC.html
http://www.reachdevices.com/Protein/buff_anion.html
I think you can get away with using sulfonate buffers like HEPES and MOPS as long as you keep the concentration down far enough, say 25 mM, and your protein binds well. Or use Tris at pH 7.5.
Since the intended pH is larger than the pI of the protein, the protein will have a net negative charge. The use of a positively charged functional group like DEAE on the resin is therefore correct.
As buffering ion you want to choose one that doesn't bind to the column, in this case therefore a positive one. Otherwise, buffering capacity and pH of the buffer would fluctuate during the run. The pH should be at least 1 pH-unit larger than the pI of the protein, to ensure sufficient charge for binding to the column. It also prevents protein precipitation (solubility of proteins is lowest near the pI). The pKa of the buffering ion should be near (within +/- 1 pH unit) of the pH, to ensure sufficient buffering capacity. Piperazine at pH 6.1 meets these conditions (there is little point to give pH with more than 1 figure after the decimal, as it depends on temperature, concentration of buffer and ionic strength). While developing a purification protocol, you can play with the pH: bringing it closer to the pI will weaken binding of the protein to the column (protein elutes at lower [salt]) and vice versa. Another aspect is, of course, the stability of your protein, which is pH-dependent.
Gravity flow can be used unless you use a high resolution gel with small beads, as they have too much back-pressure. The volume of the column determines the protein binding capacity. In IEC you want short, thick columns. Buffer, column and fraction collector should be kept cold. In addition, you have to think about protease inhibitors.
Good luck!
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