I am working on purifica of pectinmethylesterase enzyme. Many scientists have used mostly affinity chrom. For the same. I cannot find any paper where purification is done by ion exchange chrom. If I also have to perform affinity chromatography I am worried about the cost. Please guide me in this regard.
Affinity chromatography (on column) permits scientist to obtain enzyme purification at a relatively high degree with a good recovery. Generally, loss of enzyme is minimal. This is a better purification technique than those the classical ones, comprising ion exchange chromatography. Finally, what is it the scientist’s objective? It is not the technique nature, but it is purification with a minimal loss of the concerned enzyme. When the resin for affinity chromatography is not available (which is the case in several developing countries) and at all, ion exchange and other classical techniques of protein purification can be assayed.
Affinity chromatography (on column) permits scientist to obtain enzyme purification at a relatively high degree with a good recovery. Generally, loss of enzyme is minimal. This is a better purification technique than those the classical ones, comprising ion exchange chromatography. Finally, what is it the scientist’s objective? It is not the technique nature, but it is purification with a minimal loss of the concerned enzyme. When the resin for affinity chromatography is not available (which is the case in several developing countries) and at all, ion exchange and other classical techniques of protein purification can be assayed.
As long as you have an assay that allows you to test each fraction for the enzyme activity as you proceed with the purification steps, you can use ion exchange and other chromatography steps (e.g. gel filtration, hydrophobic interaction) to attempt a purification. Save the expensive affinity resin for the last step, if you still need it, and regenerate the resin for reuse. Consider also whether you might be able to make the affinity resin yourself to save money.
If possible, consider cloning the gene for the enzyme into a vector with a His-tag, expressing it in E. coli, and purifying it with immobilized metal affinity chromatography.
I agree with the above statements, with an addition. First, affinity chromatography is far faster and cheaper, assuming you have an antibody and the protein is fairly clean (ie you cannot use homogenized insect lysate without destroying the column, but supernatant from a cho cell culture has no problems). Ion chromatography usually isn't as pure as affinity and takes more optimization, don't be surprised if you need 2-4 different ion columns to get the same results as one affinity. This is simply because running one affinity column and getting >90%purify is more efficient than running 2-3 ion columns and getting the same purity. Also keep in mid that ion chromatography will take quite a bit of optimization, while affinity is usually fairly standard.
@Daniel
well, affinity doesn't have to be with antibodies only. And if you use 4 ionex columns in a row, you cannot really expect such different behaviour of the enzymes.
@Mugdha
In these articles they use only ionex columns as well. It's OK to use ionex which can give like 15-fold purification fold and then continue with e.g. gel filtration.
http://www.ncbi.nlm.nih.gov/pubmed/23887498
http://www.ncbi.nlm.nih.gov/pubmed/22512653
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