Dear researcher see this paper

Liquid-Exfoliated Mesostructured Collagen from the Bovine Achilles Tendon as Building Blocks of Collagen Membranes

suggestion by Arash sounds good. I read the paper, it is quite a procedure. You need to follow many steps.

Dear Arash Abdolmaleki ,

Indeed according to Sigma https://www.sigmaaldrich.com/NL/en/product/sigma/c9879 where they state “Reconstitution: This product is an insoluble collagen preparation. It is insoluble in water, aqueous buffers, dilute acid, and organic solvents. For use as a substrate in collagenase assays, this collagen can be prepared as a suspension in 50 mM TES buffer, pH 7.4 with 0.36 mM calcium chloride.” your observation is expected, you get a suspension (definition: “a suspension is a heterogeneous mixture of a fluid that contains solid particles sufficiently large for sedimentation.” https://en.wikipedia.org/wiki/Suspension_(chemistry)).

The paper "Pei, Y., Jordan, K. E., Xiang, N., Parker, R. N., Mu, X., Zhang, L., ... & Kaplan, D. L. (2021). Liquid-Exfoliated Mesostructured Collagen from the Bovine Achilles Tendon as Building Blocks of Collagen Membranes. ACS Applied Materials & Interfaces, 13(2), 3186-3198" (see enclosed file) as suggested by Arash Abdolmaleki looks promising but do keep in mind that they present a method to obtain collagen membranes and not a monomeric collagen solution (which is most likely with this type of collagen not possible).

However, you state that you want to perform hydroxyproline assay analysis where there is always a hydrolysis step (which makes insoluble material soluble). See for a possible good assay: da Silva, C. M. L., Spinelli, E., & Rodrigues, S. V. (2015). Fast and sensitive collagen quantification by alkaline hydrolysis/hydroxyproline assay. Food Chemistry, 173, 619-623 (See enclosed file).

Best regards.

PS. Personally I would just try your assay and see how well you can reproduce the amount as indicated by the analysis certificate provided by Sigma.