• Ensure all kit components and samples are brought to room temperature (18-25°C) before use. Thoroughly dissolve and mix all components before utilizing the kit.
• If the entire kit will not be used at once, only remove the necessary strips and reagents for the current experiment. Store the remaining strips and reagents as directed.
• Dilute the 25× Wash Buffer into 1× Wash Buffer with double distilled water.

First, centrifuge the Standard at 1000 x g for 1 minute. Then, reconstitute the Standard by adding 1.0 ml of Standard Diluent Buffer. Allow the mixture to sit for approximately 10 minutes at room temperature, and gently shake it (avoiding foaming).

Prepare 7 tubes with 0.5 ml of Standard Diluent Buffer in each and create a series of dilutions using the Diluted Standard. Remember to thoroughly mix each tube before transferring the solution to the next tube by pipetting the solution up and down several times. Set up 7 points of Diluted Standard, decreasing the concentration each time, and use a Blank with no standard for comparison. It's important to use a new Standard Solution for each experiment to ensure accurate results. When diluting the Standard from high concentration to low concentration, replace the pipette tip for each dilution. Additionally, make sure not to transfer solution into the Blank tube from the previous one.

• Before using the stock Biotinylated Antibody and Streptavidin-HRP, give them a quick spin. Dilute them to the working concentration 100-fold with Biotinylated Antibody Diluent and HRP Diluent.
• When using the TMB Substrate Solution, be sure to aspirate the required amount with sterilized tips and avoid returning any leftover solution to the vial.

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