Hi all,
I have high levels of protein loss on PD-10 desalting column (gavity flow desalting column) when trying to buffer exchange. According to GE, the recovery rate should be higher that 70%, but my recovery rate is around 50-60%. I have realised that the protein recovery is especially poor with low-concentrated proteins (< 0.5 g/L). Is that normal?
Can you please share your experience and thoughts, and/or sugest alternativies for buffer exchange of low-concentrated proteins?
Dear Ana
which protein volume did you load in the PD-10?
How did you monitor the elution?
Generally i'm loading maximun 2ml and i'm monitoring the elution with bradford reagent and in the most of cases i observed very good recovery (>80%). In few cases, i observed strong protein loss that probably was associated to protein precipitation in the coloumn (the protein elution is slow and long) probably due to incompatibility of the protein with the new buffer or stress induced to change in pH or ionic strength.
You can found a example of my procedure with PD-10 on my blog PRoteoCool https://proteocool.blogspot.com/
ProteoCool n°11(Rapid buffer Exchange by PD-10) at page 3.
i hope that it could be usefull to you.
good luck
Manuele
Hi Manuele,
I load 2.5mL and usually elute in 3.0mL.
I am working with fluorescent proteins so it is easy for me to track my sample in all steps
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