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HOME> Products> Cell Culture> Imaging dye> BindCOL, biotin-conjugated | Funakoshi

High Sensitive Denatured-Collagen Detection Reagent BindCOL, biotin-conjugated | Funakoshi

Date：November 19 2020Web Page No：95002

Funakoshi Co.,Ltd.

BindCOL, biotin-conjugated is a cyclic peptide reagent designed for the simple and highly sensitive detection of denatured collagen - an emerging disease marker. Thanks to its biotin label, detection is facilitated using various avidin or streptavidin-based systems. Unlike antibodies, this reagent specifically recognizes only denatured collagen, making it invaluable for research in collagen physiology and pathology.
It can also detect single-stranded collagen during the intracellular biosynthesis process. Please check ☞ here for more details.

Denatured Collagen Detection Reagent: DCDR

Background
Mechanism
Features
Application
Detection of single-chain collagen in biosynthetic process
Product information

Background

Collagen is a major component of the extracellular matrix and the most abundant protein in mammals. Its structure features repeated Gly‑Pro‑Hyp (4‑hydroxyproline) sequences, forming a triple-helical conformation. In humans, 27 collagen family members have been identified. Under proteolytic degradation or mechanical stress, the triple helix may unwind, producing denatured collagen (dCOL), which serves as a valuable biomarker in diverse pathological conditions, including tissue injury, osteoarthritis, fibrosis, inflammation, developmental processes, and aging.
Although previous methods using collagen antibodies could not distinguish between native and denatured forms, BindCOL’s "strained cyclic peptide" technology enables highly sensitive and specific detection of denatured collagen.

Diagram of denatured collagen — Diagram of Denatured Collagen

Advantage

BindCOL stands out due to its use of strained cyclic peptide (scCMP) technology. Unlike conventional single-stranded collagen-mimetic peptides (ssCMPs) that tend to self-assemble into nonfunctional homo-trimers—necessitating a pre-heating step—BindCOL’s cyclic design, with varied backbone lengths, maintains a twisted structure that prevents self-association. This enhances binding affinity dramatically, enabling high-sensitivity detection without pre-heating.

Comparison of single-stranded and strained cyclic CMPs

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Mechanism

scCMP consists of two peptides with different length and biotin.

Reference

Takita, K. K., Fujii, K. K., Ishii, K., Koide, T., Org. Biomol. Chem., 17, 7380～7387 (2019).
Structural optimization of cyclic peptides that efficiently detect denatured collagen.
Takita, K. K., Fujii, K. K. Kadonosono, T., Masuda, R., Koide, T., ChemBioChem., 19, 1613～1617 (2018).
Cyclic Peptides for Efficient Detection of Collagen.

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Features

Selective Binding: Targets only denatured collagen, not native triple helix
High Sensitivity: Superior performance to ssCMP-based reagents, even without heating
Versatile Detection: Compatible with avidin/streptavidin detection systems (fluorescent or HRP)

Comparison of binding affinity with conventional reagent

Comparison of Binding Ability of Single-Chain Peptides and Strained Cyclic Peptides

The binding ability of biotin-labeled existing reagents (single-chain peptides) to denatured collagen with BindCOL (distorted cyclic peptides) was evaluated. The effects of prior heating (95℃, 5 minutes) on the binding ability of both reagents were also examined. Different concentrations of each reagent were added to a plate coated with heat-denatured collagen, and the plate was detected with streptavidin-HRP/chromogenic substrate. Binding of existing reagents was observed only when heated, but BindCOL showed binding ability even without prior heating. Even when not heated, the cyclic peptide showed approximately 10 times the binding ability of existing reagents when heated.

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Application

Detection of denatured collagens derived from cultured cells

Mouse fibroblasts (MEF) were cultured for 3 days at confluence to produce collagen, and then PBS heated to 95℃ was added to denature the collagen (Hereinafter referred to as denaturation processing). The cells were fixed with 4% PFA, blocked with BSA, and incubated with BindCOL at 3 μg/ml for 1 hour. FITC-labeled streptavidin was added as a detection system, reacted for 1 hour, washed, and observed with a confocal microscope. No fluorescence signal was detected in the sample without denaturation treatment (native collagen), but predominant fluorescence signal was detected in the sample with denaturation treatment (denatured collagen).

Detection of collagen members by Western Blotting

Purified collagens (collagen I-V) were dissolved in a protein mixture solution, electrophoresed at 30 ng/lane, and transferred onto a nitrocellulose membrane. After blocking with skim milk solution, the mixture was reacted with 5 μg/ml of BindCOL for 1 hour, and detected with HRP-labeled streptavidin. Unlike conventional antibodies, this reagent can detect any type I-V collagen with different amino acid sequences.

＊ Notes to the data

Collagen I is formed as a heterotrimer from two α1 (I) chains and one α2 chain.
Collagen II is formed as a homotrimer from three α1 (II) chains.
Collagen III is formed as a homotrimer from three α1 (III) chains.
Collagen IV is formed as a heterotrimer from three of the six subunits, α1 (IV), α2 (IV), α3 (IV), α4 (IV), α5 (IV), and α6 (IV) chains, so the abundance of each subunit is low and the signal appears weak.
Collagen V is formed as a heterotrimer from three α1 (V), α2 (V), and α3 (V) chains.

Detection of total collagen in tissue and validation of its specificity

Frozen mouse tissues (liver, kidney, brain, and spleen) were broken with dounce-type homogenizer, reacted with or without collagenase for 2 hours, and solubilized with SDS buffer under denaturing conditions. Each sample was separated by SDS-PAGE, transferred onto PVDF membrane, and blocked with 3% BSA/TBST solution. BindCOL (0.4 μg/ml in TBST) was added for 1 hour, the membrane was washed, and Streptavidin-HRP (0.05 μg/ml in TBST) was added for 1 hour. The membrane was washed and detected with ECL substrate.
Collagen content varied among tissues, but bands were detected in all tissues. These bands disappeared after collagenase treatment, indicating that the reagent detects the collagen family.

＊In kidney, some bands remained after collagenase treatment, but the band position shifted, indicating that they were partially digested by collagenase.

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Detection of single-chain collagen in biosynthetic process

Collagen biosynthesis takes place in the endoplasmic reticulum (ER), where it is synthesized as a single-stranded polypeptide, and then a triple helix is formed by collagen-specific chaperone HSP47 (procollagen). It is then secreted to the extracellular space via the Golgi apparatus, where it becomes mature collagen. It has been suggested that the accumulation of immature collagen in the endoplasmic reticulum (ER) due to deficiency/dysfunction of chaperone proteins or abnormal secretory pathway leads to disease. BindCOL can detect not only denatured collagen in the extracellular matrix, but also collagen in the biosynthetic process in the ER by using the same protocol as immunocytochemical staining. It is expected to be an excellent method for detecting single-chain collagen in the biosynthetic process, such as comparing the accumulation of abnormal collagen in normal and diseased cells.

Example of detection of single-chain collagen in cellular biosynthetic process

Fibroblasts from HSP47 deficient mice and normal fibroblasts were fixed and permeabilized. BindCOL was added at 3 μg/ml and incubated for 1 hour. FITC-labeled streptavidin was added as a detection system, and the cells were allowed to react for 1 hour. After washing, the cells were observed by confocal microscopy. Remarkable accumulation of immature collagen was observed in HSP47 deficient cells compared with normal fibroblasts. By using this reagent, the relative comparison of single-stranded collagen in the biosynthetic process of each cell is possible.

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Product information

[Date : September 08 2025 00:07]

Detail Product Name Product Code Supplier Size Price

BindCOL, biotin-conjugated, Denatured Collagen Detection Reagent
DatasheetThis may not be the latest data sheet. FDV-0035 FNAFunakoshi Co.,Ltd. 60 µg $400

Contact

Description	Denatured Collagen Detection Reagent (DCDR) is high sensitive and specific detection reagent for denatured collagen which is considered as novel pathological markers
Storage	-20°C	CAS
Link

[Date : September 08 2025 00:07]

BindCOL, biotin-conjugated, Denatured Collagen Detection Reagent

DatasheetThis may not be the latest data sheet.

Product Code: FDV-0035
Supplier: FNA
Size: 60µg
Price: $400

Description	Denatured Collagen Detection Reagent (DCDR) is high sensitive and specific detection reagent for denatured collagen which is considered as novel pathological markers
Storage	-20°C	CAS
Link

Contact

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