About gel exlusion chromatography - (May/08/2007 )
Why we usually put the gel filtration column at the end of purification steps instead of earlier stages?
What is the function of this arrangement?
What is the function of this arrangement?
Could you give some more infos about "earlier stages", please. There is no standard protocol for these purifications, several protocols with different steps for different protein-families or isoforms... so some more details about your arrangement, please!
What is the function of this arrangement?
in my purifications, I often used gel filtartion at the end of purification protocol since I combined it with determination of molecular mass; this is not to recommend for crude fractions as some more protein-protein interactions may disturb determination of mass; if you like to do this, you have to calibrate your column...
the final gel filtration step is used to determine and ensure purity of the protein, you will be separating yours from other proteins of different molecular weight.
the step is also often used to put your protein into the final buffer for use or storage.
the step is also often used to put your protein into the final buffer for use or storage.
About ensurance of protein purity by GF I think it is a big question! It depends on borders of needed purity. Recently I purified a protein - urine trypsin inhibitor and apply purified fraction on FPLC (Superdex) / HPLC( Biosep) GF ( in optimal Mw separation range) for polishing and determine final purity. I 've got single Gause symmetry peak and was happy. Next day I decide to analyse purified protein on RPC18 Symmetry 300 column. When I applied slightly sloping ACCN gradient smile leave my face I saw distinct comb. Where is boards of purity??? So I decide that extent of protein purity depends on tasks. IF you need high purity only path - one band on IEF and silver staining PAAG.isn't it?
The best reason for using gel filtration at the end of a purification strategy is because unlike reverse phase, ion exchange or hydrophobic interaction (where you can load a large volume and elute in a much smaller volume), with GF you are severely limited in the volume you can load, and you always end up with a larger volume at the end. If you had to process 500 ml of starting material on a Superose or Sepharose column, you'd be forever doing that step, and you'd end up with about a litre or more. Add to that 'The Bearer's' comments about protein:protein interactions and the clean-up you'd have to do on the column every few runs, and you can see why GF is left until the end.