Dear collegues,
I am recently struggling with the dot blots I am using for quantification of peptide conjugated to polymer nanoparticles.
I have this fluorescently labelled peptide, which is conjugated to ~100nm polymer nanogels and after blotting You can see heavy coffee ring on the blot.
Did You ever come across such a problem? I am blotting 5uL of solution and real diameter of the dot is around 5 mm.
May I also add, I used to perform those blots with magentic nanoparticles conjugated with peptide and it wasn't such an issue.
(the other dot is the supernatant after conjugation, just don't pay attention to it)
Thank You in advance!
BTW: For a precise quantification of your peptides, amino acid analysis (AAA) after acidic hydrolysis is preferable. Dot blots will give you only a quite crude estimate. The coffee ring effect is not your most relevant problem...
To update You - I finally gave up the dot blot and used BCA assay instead to quantify my peptide. It seems pretty acqurate, it's well acknowledged in literature and pretty straight forward in operation.
Nevertheless, thank You for all Your answers and tips!
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