Hi,
We are running and validating some interactions of proteins via running them over a size exclusion column. We want to switch to a smaller one in order to save protein. Currently, we have a Superdex 200 16/600 pg, but it just uses up too much of our samples.
Are there any recommendations/experiences/suggestions what small SECs to use for an FPLC system (we use the NGC system from Bio-Rad). Would we run into any problems?
Just the delay volume on the NGC is 1/3 of my small SEC if I would use like a 2.4ml SEC (Superdex 75 Increase 3.2/300) or a 3ml one (Superdex 75 Increase 5/150 GL).
And what about the resolution? Does loading 50 ul on this big system with such a small SEC column influence my resolution of my protein/complex?
Additionally, I am not sure about the fractionation size and how accurate the bigger systems (FPLC) are concerning fractionation: Lets assume I use an 2.4ml SEC and fractionate 25ul fractions into a 96-well plate. I'm afraid the FPLC is not capable of doing so?
For now, I will test such a small column on a HPLC which is well suited for such low volumes of sample AND column. But we need to use a facility for the HPLC and would like to get a small SEC going on our own FPLC system. Is it possible?
Thanks in advance!
Cheers
Philipp Czermak
When you change size of column (d-diameter) or scale up, use next formula:
v1/v2=(d1/d2)2 , v-flow ml/min
and don't forget to change quantity of sample
My best regards,Antonina
Thx for the answer and sorry for my late reply. We solved the issue by simply using an HPLC :)
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