I have a 28kD protein and am trying to get rid of a 10-12kD contaminant using sephadex-25 medium. I was wondering the column size, volume and buffers that would be best for this since I am very new to the protein world. The protein has a His tag and GST and we use Ni column for purification but the contaminant is still present. I would like to make my own column to get rid of the contaminant but do not know where to start.
Give it a shot at using a different metal ion such as Co(II) or Zn(II) to see if the contaminant is not bound. If still happens do as the previous answer say.
It should be possible to separate a 28 kDa protein from a 10-12 kDa protein by gel filtration chromatography, assuming that both proteins are monomeric. Here is a handbook about gel filtration chromatography.
Hi Amy,
As Kristian said, it could be that your contaminant have aminoacid structure compatible with retention on Ni column. I understand that you have both His and GST tags. So you should try purification through the GST tag.
Gel filtration suggested by Adam is also a solution, since it induce quite important dilution of your sample.
Good luck
Gel filtration could work, but not using Sephadex G25. That is for desalting. Try a Superdex 75 column if you have one. Did you try ion exchange? That would be my choice over gel filtration. Or since you have two tag - did you try a second step with GST affinity?
Another thing is to ensure your 10-12kD contaminant has any kind of interaction/binding with your protein of interest in the separating conditions you use (ionic stenght is important). If so you could consider addition of a soft detergent in your buffer.
To preserve sample, you can try ion exchange chromatography on a very small scale with ion exchange spin columns. if it works, you can scale up.
You should first try using GST based separation as suggested by all above.however if the two proteins do not bind each other, gel filtration using a sephadex G-75 column will work well.for this you will have to use at least a 100 ml column or more to resolve them.and collect fractions of 0.5 ml which takes time as well as the sample gets diluted.If you know the charge on the two,better try ion exchanger using a pH such that one remains bound and other is in wash/unbound fraction.
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