Hey
I'm trying to measure the concentration of a protein I've purified and then labeled with Atto 647 flourescent dye.
The protein has no tryptophans or tyrosines (although does have phenylalanines) so the absorbance is very low at 280nm. I can get around this using the Bradford assay to work out protein concentration when the protein is unlabeled. However I want to label the protein with an Atto 647 flourescent dye, but can not think of a way to calculate the dye to protein ratio as there is no absorbance at 280nm as mentioned. I'm pretty sure Bradford or BCA assays will not be useful either as the solution is blue in colour because of the 647 dye.
Anybody got any tips?
Cheers!
Hi there,
UV absorbance at 205nm! As it quantifies peptide bond content of the protein. Therefore the signal is very strong and sample has to be diluted prior measurement (around 50µg/mL).
In the joint leaflet you'll find how to proceed:
I think you could still use the Bradford assay to measure the protein concentration. The extra absorbance at 595 nm from the Atto 647 dye will most likely be negligible. You will need a standard protein to make the standard curve. I recommend using one with a similar unusual amino acid composition to your protein.
It may be possible to measure the dye to protein ratio by a completely different method: intact protein mass spectrometry. The mass of the unlabeled protein is first measured. The mass, or distribution of masses, of the labeled protein is then measured. The additional mass, or masses, then tell you how much label is attached. This method is more informative than the absorbance method because it shows you the distribution of adducts, not just the average.
For detecting the presence of peptide bonds, a biuret assay would be suitable. It will not interest whether the protein includes tryptophan and tyrosines or not.
Hi there,
UV absorbance at 205nm! As it quantifies peptide bond content of the protein. Therefore the signal is very strong and sample has to be diluted prior measurement (around 50µg/mL).
In the joint leaflet you'll find how to proceed:
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