collagen type I extraction - (May/20/2007 )

I have been trying to measure type I collagen concentration in fibroblast cell culture using ELISA .
I used a kit from Chondrex. but i have never detected the collagen.
In the collagen extracting protocol provided with the kit , they use acid base extraction method. but in the end, after neutralizing with NaOH , they say to centrifuge and collect the supernatant for the ELISA . now after some searching am wondering if the collagen is precipitated so is actually in the pellet not in the supernatant.
anybody with experience with collagen extraction i have been working on this for a year now.

thanks

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Hi,
I am also wondering about collagen. I posted here several days ago, and unfortunatly got only one teply. I was going to mesure collagen with Sirius Red F3BA. There is a paper in Analitical Biochemistry 203, 187-190 (1992) called "Microplate Reader-Based Quantitation of Collagens". They describe, how to mesure collagen in fibroblast cell culture.
My problem, that I am working with tissues (very small amount) and not sure how to proceed with the extraction.
Maria

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yes this collagen is so problematic....
in the kit am using from "chondrex" they have protocol for collagen extraction from both tissue and cell culture is available on the net. mmm, but which type of collagen are you interested in.? anyway i think you can use that kit then you can precipitate the collagen of interest but adding different concentration of NaCl.
i have been told about the sirius red, and actually i spent all the day yesterday looking for the product but couldn't find a suitable one.. i will perform sirius red/fast green method but i don't think that's is enough to measure the exact concentration of collagen type I in my sample.

Louay

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