Human VWF / von Willebrand Factor ELISA Kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
-Bring all reagents and samples to room temperature (18–25 °C) before use.	-Wash by filling each well with Wash Buffer (300 µl) using a multi-channel Pipette or autowasher. Complete removal of liquid at each step is essential to good performance. After the last wash, remove any remaining Wash Buffer by aspirating or decanting. Invert the plate and blot it against clean paper towels. -Read at 450 nm immediately after adding STOP solution.

Upstream tips
-Bring all reagents and samples to room temperature (18–25 °C) before use.
Protocol tips
-Wash by filling each well with Wash Buffer (300 µl) using a multi-channel Pipette or autowasher. Complete removal of liquid at each step is essential to good performance. After the last wash, remove any remaining Wash Buffer by aspirating or decanting. Invert the plate and blot it against clean paper towels. -Read at 450 nm immediately after adding STOP solution.

Publication protocol

VWF antigen (VWF:Ag), VWF–HA and VWF–MYC protein levels in conditioned medium and cell lysates were measured by ELISA. For VWF:Ag measurements, ELISA plates (Greiner, Frickenhausen, Germany) were coated overnight with polyclonal antibody rabbit anti‐hVWF (A0082; Dako, Glostrup, Denmark) diluted in coating buffer (100 mm bicarbonate, 500 mm NaCl, pH 9.0). Samples were diluted in wash buffer (50 mm triethanolamine, 100 mm NaCl, 10 mm EDTA, 0.1% Tween‐20), and incubated for 2 h. Polyclonal antibody rabbit anti‐hVWF coupled to horseradish peroxidase (HRP) (P0226; Dako) was used as the detecting antibody, and diluted in wash buffer. Wells were incubated with secondary antibody for 2 h. O‐phenylenediamine dihydrochloride (OPD) (Sigma‐Aldrich) was used as substrate, and one tablet of OPD was dissolved in 24 mL of substrate buffer (22 mm citric acid, 51 mm phosphate, pH 5.0) plus 12 μL of 30% H2O2. Wells were incubated with substrate solution, and the reaction was terminated by the addition of 2 m H2SO4 after 15 min. Normal pooled plasma (NPP) was used as the reference.

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Manufacturer protocol

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