Hey, I am urgently looking for people that use the EZQ protein quantitation assay. It is used because it is one of the quick and easy methods to determine protein content with reduced interference with polyphenolic compounds. However, in my case I am using i.e. cranberry juice, and the anthocyanins seem to get stuck, together with the protein, on the membrane and won't completely wash off during the methanol wash step.
So I am looking for people who have similar experience with this assay and may also use pigmented samples that precipitate onto the membrane.
A tech from the vendor who sells this product said that anthocyanins may interfere if they precipitated with the protein on the membrane and don't wash off, it depends on if the anthocyanins are able to fluoresce at the excitation/ emission wavelengths used (and interfere with the EZQ reagent).
I looked up some articles but it seems that the wavelengths used for fluorescence are out of range for anthocyanins, so I am wondering if not that, if the anthocyanins (and maybe other compounds) get stuck with the proteins on the membrane and sort of block a correct treatment with the fluorescence reagent and/or affect the reading of the plate, which would maybe mean that the readings are lower than they should be?
Any ideas?
The interference is likely to be fluorescence quenching via pi pi interactions with anthrocyanins. This is also likely extra bonding interaction between analyte and interference, responsible for them remaining intimately together through extraction and membrane binding. I do not have a direct solution, but this might put you closer to a workaround.
Good luck!
- Badges
- Science topic
- Similar topics
- Chemistry
- Biochemistry