I conjugated some antibodies to oligos for CITE-seq.
I need to determine the final concentration of my antibodies.
Using the NanodropOne's Protein A280 detects that there is DNA contamination (as expected), and offers a corrected measurement taking this into account.
Does anyone know if that corrected measurement is reliable, or should I run a different type of assay?
DNA is measured at 280 nm using a Nanodrop One device. Protein is detected at 260 nm and salt at 230 nm. Readings would be provided for each category. Make sure to only confirm your 260 reading since that is the sample area of interest you are measuring.
First, I assume that you have removed free oligo from the conjugate? Unless that separation is efficient, you will overestimate the oligo regardless of how you try to do the measurements.
Second, the A280 reading of the Ab is 'polluted' with oligo signal which is still significant at A280, despite it being maximal at A260. Equally, the A260 signal for oligo has additional signal at A260 attributable to the Ab.
This is considerably more complicated than say with organic dyes, where you can clearly separate the dye signal at a much higher wavelength (e.g. A488), and then determine the relative contribution of dye at A280 by measuring dye alone at the two wavelengths (A488 and A280), which affords a correction factor for the dye (i.e. allowing you to calculate the excess signal at A280 caused by dye).
In the oligo-Ab conjugate, as you know neither the concentration of the Ab nor the concentration of the oligo, I do not see how any valid correction can be made from the analysis of the conjugate ALONE.
You would need to understand, in addition, the absorbance profiles of Ab and oligo alone, and apply the kind of mathematical treatment used in flow cytometry ('compensation'), where dyes can reciprocally spill over into the 'wrong' channels.
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