Hello,
I got a question about optimizing my FPLC separation for my 55 kd protein from a pre-attached solubility tag of 30 kd protein.
I am using 6x his tag and tev cleavage. After doing the Nickel column, I still got some of the 30 kd solubility tag in my sample and for this I use FPLC to get rid of it.
However, I am getting poor separation of the TAG and I end up with both my target protein and the solubility tag in the same fraction. Also, it sounds like my protein comes out in two fractions instead of one (10,11) as confirmed by SDS page.
I need some advice or articles recommendation for optimizing this separation to get rid of the solubility tag and also to get most of my target protein in one fraction.
Following are all the parameters I am using for the FPLC separation.
Column type: Superdex 200 increase 10/300 GL
Column volume: 23.562 ml
pressure limit pre-column: 2.6 MPa
Flow rate: 0.4 ml/min
Loop type: capillary loop
Isocratic elution volume: 30 ml
Fixed fractionation volume: 1 ml
- Start fractionation after 5 ml
It would help to add the name of your solubility tag instead of keeping it secret. If you are working with GST as the solubility tag you should know that it comes with a few issues. GST tends to homo-dimerize so you might get a dimer with one (or two) His-tags that are not accessible to the Ni-beads and could not be separated by Ni after the cleavage. So you have a dimer with ~60 kDa that elutes in the same range as your 50 kDa target protein. Another issue with GST is that it tends to be less soluble when you have a His-tag at the N-terminus, in most comparisons His-GST shows the lowest performance in terms of solubility improvement compared to e.g. GST alone. Instead of His-column you could use a GST-column to remove the cleaved GST before SEC.
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