No, this assay does not measure hydroxyproline. This assay quantifies N-terminal glycine containing peptides from an enzyme based degradation of collagen. We have a hydroxyproline assay, DHYP-100.
You may also wish to incubate the digest mix step for an extra 30 minutes at 37°C (90 minutes total) to ensure that the digest reaction is complete.
Fluorescence is measured differently depending on the plate reader. This is why plate readers use relative fluorescent units (RFUs) to quantitate the fluorescence readings. These are arbitrary units to assess the fluorescence intensity of the wells relative to each other. The numbers you see on the standard curve in the protocol are from the assay being performed on our lab’s plate readers. It is completely normal for your assay to produce different fluorescent readings; what is more important is to focus on the linearity of your standard curve.
Either your samples are diluted too much, or have not been diluted enough. We recommend running a serial dilution on your sample to determine the optimal dilution factor for quantification. This is the range at which dilutions start producing similar collagen concentration estimates after being corrected for dilution factor.
Yes, this assay can be used on store bought collagen. Because the collagen is often very concentrated, you will likely need to dilute it greatly (e.g.100-1000 fold) for it to be within the assay’s detection range.
Hong, S., et al. (2020). Ginsenoside compound-Mc1 attenuates oxidative stress and apoptosis in cardiomyocytes through an AMP-activated protein kinase-dependent mechanism.Journal of Ginseng Research.44(4): 664-671. Assay: Collagen in mouse heart tissue.
To find more recent publications, please click here.
If you or your labs do not have the equipment or scientists necessary to run this assay, BioAssay Systems can perform the service for you.
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Please email or call 1-510-782-9988 x 2 to discuss your projects.
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