We know the capacity of the gel and the total amount of protein in the bacterial lysate from a
Bradford assay. What we do not know is what fraction of the total protein is the his-tagged protein of interest. We also do not know how much excess capacity is considered a good choice. For example, if one knew that there were 100 mg of His-tagged protein and that having a twofold excess were a good choice, one could choose the volume that produced 200 mg of capacity.
Dear Chris
in my experience with Nickel, is good use an amount of resin to be close to the saturation and it is not important the total protein but the amount of your specific protein to improve the purity level.
Generally i'm performing a small scale expression and purification trial using 100ul or little more of resin to have an idea of the productivity in terms of mg of target protein/ml of culture and on this basis of this results i'm calculating the resin volume of the scale up considering the real resin binding capability about 50-60% the one reported in the resin supplier datasheet (but t may change a lot on the basis of protein MW, for example if you have protein with MW > 100KDa the excess the protein binding efficiency may decrase due to protein steric hydrance that may cover some resin binding site)
ciao
Manuele
I agree with Manuele Martinelli. It is necessary to do a small-scale expression and purification experiment, and the volume of the resin I use is also estimated based on preliminary experimental results.
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