Dear all, I have a protein (10Kda) which can form a complex with another ligand(400Da). The ligand is not soluble in water. It can be soluble in DMSO. I would like to check the complex protein-ligand formation in size exclusion columns. Whenever I add my protein to the ligand or vice versa, it gets precipitated (almost tried in different buffers) and most of them will stay in the column and it won't elute at all. Moreover my protein doesn't have tryptophan residue which could absorb UV. My ligand can observe 230 nm wavelength. Is there a way I could check the complex formation in a Gel filtration column? And is it possible to do a multiwavelength UV absorbance in AKTA FPLC.
Really appreciated all your suggestions.
have u tried gradient elution mode...?? what is ur elution buffer..what is the kind of resin u r using...??
I'll get back to you soon...its easy to do gradient mode elution..try to do it with sephacryl resin....also let me know what is volume of the column...I'll give you a protocol considering all these....
Oh k...I'll get back to you soon...I'm trying to give you the best possible option...
Yes, with the AKTA FPLC, you can definitely determine the absorbance at more than one wavelength, simultaneously.
Hi! As Stephanie wrote, you can set the absorbance that you need. New Aktas can read more than one wavelenght at the same time, but some older models can read only one (so check what your machine can do).
230nm is not only the absorbance of your ligand, but also the absorbance of the peptide bond, which means that also your protein will give a signal. Before running your complex, I would suggest to run your protein and your ligand separately, so that you'll know which are their own elution volume, and you can use them as reference to check if there is any change with the complex. I don't know if Superdex 75 can appreciate a difference of 0.4kDa, it could be quite hard too distinguish the complex from the protein alone, but you can try!
Moreover I suggest you to find a proper buffer to keep both your protein and your ligand in solution, and after you found it, check if its composition is compatible with the column...Superdex are quite resistent, but there are some limits on what you can use (you can find everything in the datasheet of the column). If your protein and ligand keep precipitating in the column, the efficiency of the column will start reducing (unfortunately we experienced this situation in our lab). If you want to appreciate small shifts your column needs the maximum of its efficiency, and this means a carefull general mantainance.
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