Hello,
I have some doubts about how to interpretate the 5% breakthrough obtained during a dynamic binding capacity tests on an affinity chromatography column.
In my view there are 2 possible interpretations:
1) I reach the 5% breakthrough when the concentration of the effluent of the column (unbound) is the 5% of the concentration of the fraction I am currently loading.
2) the 5% breakthrough is reached when the whole unbound fraction contains the 5% of the total amount loaded up to that moment.
The two calculations give completely different results.
What do you think, what is the most correct approach?
Thanks a lot
Luciano Capponi
Hi Luciano capponi,
If you use purified protein without any other UV interference impurities, then it will be easy to find out 5% or 10% of breakthrough point.
If you are using chromatography system with purified protein with small volume column:
1. Dilute your purified sample to OD 1 to 1.5 (no UV saturation because of high conc).
2. Load diluted purified sample without column (column bypass) : get base line - consider 100% of achieved UV.
3. Wash sample with equilibration buffer followed by column Inline equilibration and loading at specific flow rate (residence time).
4. Initially no UV/low UV will be observed when column binding sites occupied by protein., protein will come out from column and you can observe UV. Ideally you should saturate the column and loading till 100% UV... That is called saturation point.
4. calculate the hold up and subtract from the actual volume where you are getting UV 5% of 100% UV.
5. Volume X conc = total conc. Then total conc. / resin volume will give DBC (5%) / mL of resin.
If you are using crude sample then you have to collect all fractions and the analyse the protein conc. or OD then plot breakthrough curve and find out the DBC (5%).
For DBC : loading conditions ( buffer type, operating pH, conductivity and flow rate(residence time) will important factor to consider.
Please feel free to write me for any further assistance.
Hope this helps.
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