hello thr, I am trying to purify my enzyme by using IEC (anion) using biorad FPLC unit. I tried isocratic as well as gradient elution methods but getting four flat peaks out of which first two are of my enzyme which should come in jus one fraction or peak. I used 20% isocratic flow. the elution profile is weird when i use gradient elution. please help. attaching a pic of my chromatogram
Could be possible for your enzyme being a glycoprotein with charged oligosaccharides??? (for example neuraminic acid?? or phosphate?). If you have different glycoforms of the protein with slight differences due to charged carbohydrates, then, it is difficult to obtain a sharp peak when using ion exchange chromatography.
Hi Mugdha,
You are using FPLC for protein purification, in which you get the peak for proteins based on their concentrations, so for checking the presence of enzyme you have to collect the fractions and do the enzyme activity assay. Then only you can decide in which fraction your enzyme is getting eluted.
You are using anion exchange chromatography, if your are using isocratic system, then check the enzyme activity of fractions collected at different concentration of salts (KCl or NaCl) in elution buffer. From this you will get an idea that exactly at what concentration of salt your enzyme is getting eluted and then using that data design the gradient. You will definitely get the sharp peak of the protein of interest.
@Arshad
what do you mean by isocratic system? The FPLC should have two pumps controlled by computer. In case you have LPLC, that should still have some mixer. In case you really do have only one line, you could create your mixing device by putting your first buffer into some closed beaker or something and bring in line with your elution buffer. As the buffer A will be sucked out, if the beaker is closed well enough, it should suck in the elution buffer.
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