Generally we want dyes whose spectra or brightnesses are modulated by their interaction with a species of interest. I need the opposite of that.
I am developing a 96 well microplate fluorescence assay and am plagued by irrepeatable well bias. That is, filling a plate with uniform dye shows some wells to read brighter than others, but the brightness map is not repeatable enough to create a correction factor. (image: intensity map of a plate. Relative differences from the mean plate value are scaled from -6% to 6% in blue-white-red; square sizes are scaled from absolute 0 to absolute 6%)
What I would like is to use a second dye emitting at a different wavelength from Rhodamine B as an internal standard. Since this second dye will be in solutions with different protein species, I want it to be minimally altered by their presence. Being unperturbed, its brightness would be uniform, and measured differences would manifest the instrumental artifacts and not dye behavior.
To answer questions before they are asked:
1. Don't you think your problems come from inconsistent pipetting?
It's a very reasonable thought, but rotating the plate shows pipetting does not explain all of the variance. Furthermore, I can get excellent coefficients of variation in some parts of the plate (0.99), but moving samples to a different part of the plate will unacceptably alter intensity values. Small differences in meniscus formation are likely drawing out optical artifacts.
2. Maybe the plates are bad?
It could be. I've tried several kinds, each have their own signature issue. I have one more to try before I give up on that direction.
3. Maybe just call the instrument company?
I'm working with them. The problem is they have their ways to qualify an instrument, and they don't involve real samples in real plates. Getting a good audience takes some work. I'm working on it.
4. Your artifacts may be different at different wavelengths.
In which case I would be hosed. My hope is wavelength specificity is a second order mess.
5. This internal standard plan sounds messy. Are you sure you want to go down this road?
I want the assay to work! None of my roads are well paved... I am in New Jersey.
Any suggestions? Water-soluble dyes, please.
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