buffer exchange problem - (Oct/15/2007 )

hi all,

i had successfully my protein, subsequently i want to inoculate the protein into mice. however, i have to do buffer exchange before the inoculation. is it any method to check whether my sample is probably desalt?

i had urea, imidazole in my sample, any buffer exchange column are compatible? any methods are recommended to remove these contaminant?


thanz

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Sephadex G25 is a common resin to buffer exchange technique. Concerning your protein sample with urea and imidazole. If urea concentration is near 6-8M so I think a problem will arise with desalting, deals with aggregation and precipitation of your protein in column.

If you would like to make sure about efficacy of buffer exchange so you should apply volume of your protein mixture near 5% of resin volume ( 250ul to 5 ml Sephadex G25 column) I work usually with prepacked HiTrap Desalting columns ( GE Healthcare)

If you would like to look at this process with your eyes and estimate efficacy I think you may add dye ( for ex phenol red) to your protein mixture. The front of this dye or near this contains also all your low molecular substances which you remove

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keep in mind that you still need to maintain an ionic strength over 0.15M (25mM NaCl is okay if you use sephadex) to ensure that there is no non-specific binding to the matrix (this includes dialysis membrane if you choose to dialyze instead of a column method).

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why not dialyzing? useful even at low amounts

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