Is it ok to purify protein-FITC (10 microgram) using dialysis membrane(3 kDa), protein mol wt 20 kDa? What solvent would be ideal? FITC has poor solubility in DI water. So worried that unreacted FITC may not be completely removed during dialysis.
Please suggest.
Considering the tiny amount of protein you are working with, I think it would be better to avoid any separation step, which will cause a loss of protein. Instead, when the labeling step is finished, add a sufficient concentration of a primary amine (such as Tris or ethanolamine) to consume all the remaining FITC (although it will probably have degraded anyway by then, since it is unstable in water). The unbound fluorescein will still be in the sample, but it will be unreactive. That may be OK for some applications, such as flow cytometry or immunofluorescence microscopy, wherein any free fluorescein will be washed away.
Hi Ajeesh,
In general I use dialysis for these sort of reactions and did not lose a large amount of protein (SlideALyzer cassettes work very well for that). But as Adam said - if you only have such a very small amount of protein you might es well just tolerate unreacted FITC in your mix. However, if the presence of unreacted FITC is an issue for your application you might consider FPLC size exclusion chromatography.
You may also use centrifugal ultrafiltration equipment for dialysis purposes at low volumes and low protein concentration without poor recovery. Ultradiafiltration could be applied to perform buffer exchange using a 3 kDa membrane with consecutive wash. Thereby you can concentrate your trace amount protein but also can clean-up your protein solution. You will also have the flexibility to use different solvents to extract the unreacted FITC if you carefully examine the solvent suitability & stability of the membrane you choose.
See the link below for protocol example;
https://www.merckmillipore.com/TR/tr/life-science-research/protein-sample-preparation/dialysis/amicon-ultra-diafiltration/hIyb.qB.sacAAAFADHxwFMtG,nav
Good Luck.
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