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

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.

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.

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.

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.