The source of my Glucagon was Human Glucagon from European Pharmacopoeia(EP) Reference Standard from Sigma-Aldrich; I used a few different plate conditions with (1) Standard Nunc Plates, (2) Nunc Plates with a Poly-Lysine coating and (3) the Greiner Non-binding plates.
Experimental Conditions were 0.6mg/ml Glucagon dissolved in 3.2mM HCl, 0.9%w/v NaCl with 50 microMolar Tht. There was 5s of automixing before each fluorescence measurement.
The second plates scenario worked the best in terms of getting something close to a typical sigoimdal curve but in the other scenarios I was getting some weird behaviour. Has anyone seen anything similar? Attached is a pdf of my overlaid-raw Tht assay data.
Any help would be appreciated
It turns out the Glucagon has a competing 2-pathway mechanism for the formation of fibrils. One pathway forms these temporary twisted fibrils that bind Tht and have a high fluorescence intensity. These fibrils then de-polymerize and feed into a second pathway that forms stable fibrils with that binds Tht with a lower intensity. If this is dominant the aggregation profile will show a growth phase that end with a high peak, and then the fluoresence will decrease from the maximum.
If the pathway that forms the stable fibrils is dominant, we then get an aggregation profile that looks more like a sigmoidal curve.
Of course we can aggregation profiles that are in between these if the balance of the pathways play out in different proportions.
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