Hello, i would like to use chromatographie for protein purifications and we dont know how to use sepharose Cl6B who alrealy equilibrated with buffer unknown.
Hi there,
Sepharose is made for size exclusion chromatography (SEC), not for ion exchange chromatography. If you don't know what is the composition of the equilibration buffer then pass one bed volume of water and then equilibrate with one bed volume of your equilibration/running buffer. SEC is always performed with isocratic condition (same buffer and constant elution rate) and is based on separation according to the apparent size of objects (bigger ones eluted first and smaller ones later).
Replace the buffer as Dominique says, I would use at least 3 column volumes. Are you sure you just have gel filtration medium?
I presume the resin for the ion exchange chromatography (IEC) you wish to use is DEAE-Sepharose CI6B. This is a good resin whose use is dated as far back as early 1970s (product of Pharmacia). So it will be safer to get a note from the manufacturer on the preparation of the resin.
However, for an already packed IEC column using DEAE-Sepharose CI6B, it is expedient and extremely important you re-equilibrate the column with a buffer solution of known pH and concentration the same as the condition in which your protein sample is solubilized. This same buffer should be employed as the running buffer (mobile phase) for the IEC before Increasing the concentration in stepwise elution or the addition of Increasing salt concentration (gradient elution) of bound proteins.
Note: Required buffer solutions are not chosen arbitrarily. A search through literature will help you know which buffer is needed to maintain the structural integrity and activity of your protein sample.
Sepharose CL6B is made for size exclusion chromatography (SEC), not for ion exchange chromatography. Equilibrate the column with your buffer (1.5 column vol.) and fractionate in the same buffer. MW Range for proteins 10.000 -40.000.000 D.
If you want to perform ion-exchange chromatography, select another sorbent (for example DEAE-Sepharose, CМ-Sepharose, equilibrate the column with at least 3 volumes of a column of a suitable buffer, remove the salt from the sample (dialysis) and apply the desalted sample to the column and so on.
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