Hello Peers,
Warm wishes to all. I am a new user of the Cytiva AKTA Start system for SEC with a column, HiPrep 16/60 Sephacryl S-100 HR (120 ml column volume). I am not sure about the minimum concentration of protein to be injected into the column for detectable peaks. Kindly please help me with this. Thank you in advance.
Regards
Pratibha Kushwaha, PhD
University of Hyderabad
Dear Pratibha Kushwaha
your answer do not have a simple answer since it depends from the protein properties and from the wavelenght that you use for detect the protien.
If you are using A280nm, the detection limit may change a lot from protein to protein on the basis of its extincion coefficient that is related to the % of aromatic AA (W, Y and F)
extinciton coefficent could be calculated usiing
https://web.expasy.org/protparam/
Considering that the minimun detectable signal of an istrument similar to AKTA could be considered about 10mAU and that during the run you will observe 5-10 fold sample diution, i think that the minimal concentration to be injected to the coloumn to be detectable is the one that provide on the basis of its exctinction coefficient a theorefical absorbance of al least 200mAU (0,2AU)
best regards
Manuele
I would approach this empirically. If your protein is not in short supply, inject an amount and you will know the answer. Based on the product insert, I think you should be able to see 100 micrograms of a mid-sized globular protein (e.g. albumin) from a single injection. Sometimes you just have to try. Best of luck.
https://webhome.auburn.edu/~duinedu/manuals/Sephacryl_new.pdf
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