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Hi Jim, If I remember correctly the delay volume from the pumps to the top of the column is roughly 8 ml for Äkta Purifier, Explorer and also Prime. The actual volume from the bottom of the column to the fractionator, or more important from the UV cell to the fractionator depends on the tubing and must be calulated by hand. Normally this is already done and you could find this value in the software in the fractionation part. The standard systems Explorer, Purifier starts with the fractionation after this dead volume. So you can wait roughly 8 ml plus your column volume, start the fractionation and than your protein should be in one of the first fractions, assuming a step gradient. But be cautious it is just a rough estimation!!! Christian Am Dienstag 21 Dezember 2010 16:17:13 schrieb James Pauff: > Hello all, > > Does anyone have a rough estimate (or has anyone actually determined) an > average dead/delay volume between buffers run on an AKTA Prime FPLC? We > are attempting to overexpress/isolate a smaller His-tagged transmembrane > protein, and require running several detergent buffers in succession over > the column. This obviously creates fractions that are just dead > volume/ddH2O between buffers, and we would like to narrow in on where to > look for the protein prior to analyzing the fractions (i.e. how many > fractions can we discard?). Should we run ddH2O and then the first mL > into 'waste' before running each buffer over the column (and collecting > fractions)? Just not used to this system yet! Thank you! > > Jim >