Hi there,

I guess you mean grafting different motifs on the same matrix for affinity purification purpose? Well I've never tried but for instance you could give it a go by mixing NiNTA beads and GSH beads and see if you can selectively purify His-tagged and GST-tagged proteins issued from the same sample. But I think the easier would be to perform successive affinity chromatographies using the flow through of the first as loading onto the second aso... But it's only a personal feeling.

I agree with Dominique. The best way is to connect together (in tandem)  2-3-4 columns with different specificities and recycle your protein mixture through all the columns overnight at low flow rate. Then you disconnect the columns, wash each of them with appropriate for each of them buffer, and make elution of the bound protein from each of the columns. We did it many times in our lab with different monoclonal antibodies and it always worked well. 

If you want to express entirely different proteins you may have higher yields for all the proteins using individual cultures as one protein may be transcribed & translated preferentially.

Often multiple tags are used when trying to purify a complex. A simple example would be a HisTag on one protein and StrepTag on the second. The elution from the His-Trap could be passed through a Strep-Trap to isolate a complex containing both partners.

in theory you couldprepare a series of affinity resins based on ligand binding for a series of proteins.  If you then mix the individual resins each should behave essentially independently in the column.  You could then elute the different proteinsd with their specific ligands.  This would work for different protein:ligand pairs but would obviously not be applicable ti antibody based recognition or other non-specific binding interactions.

Well, my experience is that, in general, the elution conditions (the optimal buffer compositions) are rather different for the different affinity chromatography purifications. When you have only two different tags it might be possible to do tandem purification for the two tags; however, in such case the binding and elution conditions must be rather close for the two different tags. Think twice before plan such work, that would be my suggestion.