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Denatured Collagen Detection Reagent: DCDR | Funakoshi

Date:November 19 2020Web Page No:95002

Funakoshi Co.,Ltd.


DCDR

This item is high sensitive and specific detection reagent for denatured collagen which is considered as novel pathological markers. Due to biotin-labeled-probe, it can be detected by Avidin- or Streptavidin-conjugated reagents. Also, it is compatible with unfolded or misfolded collagens in ER under the biosynthetic pathway.

What is Denatured Collagen?

Collagen is the most abundant component of the extracellular matrix (ECM). Collagen family has unique triple helical structures consisting of three polypeptides that contain frequent repeats of Gly-Pro-Hyp (4 hydroxyproline) triplets. Once triple helical structures are loosened due to heat, proteases, mechanical stress and chemical modification, the collagen becomes denatured collagen. This denatured collagen is considered as pathological markers; such as fibrosis in liver, osteoarthritis in cartilage, atheroscl erosis in blood vessels and abnormal invasion of cancer cells.

native-denatured-collagen

Detection methods for Denatured Collagen

Antibody:
Antibodies are one of the general tools for collagen research but can not distinguish between native and denatured collagens.
Only few antibodies are established to specifically detect denatured collagen, but they recognize only one specific collagen isoform.

Chemically synthesized collagen mimetic peptides (CMP):
CMPs selectively hybridize in the unfolded region of collagens. However, conventional single stranded CMPs (ssCMP) are preferentially self-assembled to homo trimer in water and it dramatically reduces binding affinity to denatured collagens. To avoid self-assembly of ssCMPs, a pre-heating step is required so that it could increase labor and experimental bias in each trial.

"New reagent" Denatured Collagen Detection Reagent:
The peptide has highly unique structure which is a strained cyclic CMPs (scCMP) and has optimized anionic charges. Due to this property, the self-assembly is suppressed. The scCMP improves not only heating step but also binding affinity for denatured collagen, over 10 fold higher than ssCMP. Our “Denatured Collagen Detecting Reagent shows dramatically higher sensitivity than conventional ssCMP without pre-heating step.

ssCMP-scCMP

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Denatured Collagen Detection Reagent

collagen-structure
scCMP consists of two peptides with different length and biotin.

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Features

-Can detect denatured collagen (and also immature condition in ER) specifically.
-Dramatically higher sensitivity than conventional reagent.
-Detected by fluorophore/HRP-conjugated avidin/streptavidin

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Experimental data


Quantitative analysis of binding affinity of both conventional ssCMP and “Denatured Collagen Detection Regent"
Wells on 96 well plate were pre-coated with heat denatured collagen I. The wells were washed by ELISA buffer (20 mM HEPES Na (pH 7.5), 100 mM N aCl, and 0.005% Tween 20) three times and blocked by 0.5% skim milk. After washing the wells, ssCMP or “Denatured Collagen Detection Reagent" containing solution were added into the wells and incubated for 1 hour. The wells were washed by ELISA buffer three times and probed with streptavidin HRP conjugated. Binding intensity was estimated with a colorimetric substrate for HRP.
collagen-data1

Detection of denatured collagens derived from cultured cells
MEF cells were cultured at confluent condition for 3 days to produce collagens. For preparation of denatured collagens the cell layers were treated with hot PBS (95℃) for 1 min . After the denaturation, they were fixed with 4% PFA and blocked by 3% BSA /PBS. They were incubated with 3 ug/ml of the reagent in PBS for 1 hour, washed by PBS three times and incubated with streptavidin FITC conjugated for 1 hour. They were washed and observed by confocal laser microscopy. The “Denatured Collagen Detection Reagent" specifically detect denatured collagen, but no signal from native collagen was observed.
collagen-data2

Detection of collagen members by Western Blotting system
Purified collagens (collagen I, II, III, IV and V) were separated by SDS PAGEand transferred to a nitrocellulose membrane. The membrane was blocked by skim milk, probed with 5ug/ml the reagent for 1 hour. After washing the membrane with TBS, the membrane was treated with streptavidin AP conjugated to detect biotin conjugated this probe. The “Denatured Collagen Detection Reagent” could detect all collagens I-V by the SDS PAGE/ Western Blotting system.
NOTE: Under SDS PAGE, all collagens were denatured. This result showed the reagent detect total collagen in the sample.
collagen-data3

Detection of intracellular unfolded and misfolded collagen
Normal MEFs and HSP47 KO MEFs were fixed with 4% PFA, permeabilized by 0.5% T ritonX 100 and blocked by 3% BSA. After washing cells, the cells were probed with the reagent (3ug/ml) for 1 hour, washed and further treated with streptavidin FITC conjugated for 1 hour. After washing cells, the cells were observed in confocal microscopy. Collagen specific chaperon HSP47 KO MEFs show higher signal from ER than normal MEFs. The “Denatured Collagen Detection is a valuable tool to estimate the amount of intracellular unfolded and misfolded collagen accumulation inside cells.
collagen-data4

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Reference

  1. 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.
  2. 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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Product information

[Date : January 26 2021 23:29]

Detail Product Name Product Code Supplier Size Price
Denatured Collagen Detection Reagent
DatasheetThis may not be the latest data sheet.
FDV-0035 FNAFunakoshi Co.,Ltd. 60 µg $360

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 : January 26 2021 23:29]

Denatured Collagen Detection Reagent


  • Product Code: FDV-0035
  • Supplier: FNA
  • Size: 60µg
  • Price: $360

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

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export@funakoshi.co.jp

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