Hi
I have a protein labeled with rhodamine B and after reaction i used Zeba spin column for dye removal. my problem is that lot of unreacted dye remained which interfered with my florescent microscopy later.
So my question is if there is a easy way to remove unreacted rhodamine? does Dialysis work? are there better dye removal spin column than Zeba desalting spin columns?
If the dye concentration is too high, a single spin column may not be sufficient to remove all of it. A second spin column may get rid of what remains after the first.
Larger spin columns, such as PD-10 columns can handle more protein and dye.
I usually uses NAP-10 coulmn for the removal of dye. Which is very effective if you have sample volume of 1-1.5 ml.But i agree with Dr Adam, you can spin your coulmn twice.Some time when overlabeling occur ,it is the best way to remove extra dye. I have also used NAP-10 coulmn twice to remove the extra dye.
Another method is using the Amicon filter (According to your protein MW) ,You can spin your sample 2-3 time untill the flowthrough didnt show any dye absorbance.You can check the flowthrough absorbance by spectrophotometer at each step.
However, this method cause protein loss at each step.
Good luck
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