VWF collagen binding activity (VWF:CB) was determined using ELISA as described [32 (link),33 (link)]. Briefly, 25 μg/mL of human collagen type III (Sigma-Aldrich, Saint-Louis, MO, USA) for binding of human VWF or 25 μg/mL of human collagen type I (Sigma-Aldrich) for binding of bovine VWF was coated onto a 96-well microtiter plate and the plate was blocked with a 3% milk powder solution. Next, a 1/80 dilution of plasma was added and the samples were further diluted ½ in PBS containing 0.3% milk. Bound VWF was detected with HRP-labelled rabbit anti-human VWF antibodies (Dako) and visualized as described above. The VWF:CB was calculated using NHP as a reference of which the VWF:CB was set at 100%.
Human collagen type 3
Manufactured by Merck Group
Sourced in United States
Human collagen type III is a laboratory product that serves as a structural component of connective tissues. It is a naturally occurring protein found in the human body. This product can be used for research purposes to study the properties and functions of collagen in various biological applications.
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Lab products found in correlation
2 protocols using human collagen type 3
1
Collagen-Binding VWF Activity Assay
2
Comparative Collagen Characterization
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human collagen type III, human collagen type I, and rat collagen type I were purchased from Sigma. According to Sigma, human collagen type I and type III was purified from placenta, and rat collagen type I from tail tendon. The purchased collagens were solubilized in 20 mM acetic acid, pH 3, at 4°C and 2.4 mg/mL. Fresh rat collagen type I was prepared from a single rat tail tendon following a procedure published by Dr. Sergey Leikin’s group, and the precipitated collagen was solubilized in 20 mM acetic acid, pH 3, at 4°C and 3.0 mg/mL [46 (link)]. Collagen was mixed with 5X SDS sample loading buffer containing 60 mM Tris-HCl pH 6.8, 25% glycerol, 2% SDS, 350 mM DTT, and 0.1% bromophenol blue, and was run on a 4–20% Precise gel or a 7.5% SDS PAGE gel and stained with coomassie blue. Bands of alpha chains were excised from the gels and submitted to The Rockefeller University for in-gel digestion and mass spectrometry analysis.
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