EnzyFluo™ Collagen Assay Kit

Protocol SDS

Application

• For quantitatve determination of collagen in biological and cosmetic products.

Key Features

• Safe. Non-radioactive assay.
• Fast and sensitive. Linear detection range (20µL sample): 2 µg/mL to 50µg/mLcollagen in 96-well plate assay.
• Convenient and high-throughput.”Add-mix-read” type assay. No wash and reagent transfer steps are involved. Can be readily automated for processing thousands of samples per day.

Method

• FL375/465nm

Samples

• Biological samples (e.g. plasma, serum, cells) and cosmetic samples (e.g. collagen solution)

Species

• All

Procedure

• 80 min

Size

• 100 tests

Detection Limit

• 2 µg/mL

Shelf Life

• 6 months

More Details

COLLAGEN is the key structural protein of connective tissue and the most abundant protein in mammals. It occurs in many different types and forms with Types I -V being the most common. Aside from the crucial role it plays in the body, it has numerous medical applications such as its use in reconstructive surgery including bone and skin grafts. It is also commonly used in cosmetics due to its anti-aging and skin healing properties. Assay methods available for quantifying collagen currently range from needing extensive hydrolysis procedures with acids and bases to using expensive antibodies and complicated protocols. BioAssay Systems collagen assay kit delivers a very simple, safe, and sensitive method to quantify collagen in samples. In the first step of this procedure, collagen in the sample is enzymatically digested into peptides. Subsequently, the N-terminal glycine containing peptides react with the dye reagent to form a fluorescent complex. The fluorescence intensity of this product, measured at λex/em = 375/465 nm, is directly proportional to collagen concentration in the sample.

Does this assay measure hydroxyproline?

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.

My standard curve isn’t turning out linear / I am getting inconsistent results?

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.

I’m not getting the same fluorescence readings for my standards as the standard curve on the protocol?

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.

My sample, which I know has collagen, isn’t measuring any collagen with this assay?

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.

Can I use this assay to measure cosmetic or beauty product collagen?

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.

– Fast turnaround
– Quality data
– Low cost

Please email or call 1-510-782-9988 x 2 to discuss your projects.