Q: How should samples be stored?

A: For long-term storage of samples, freezing at -80°C or below is recommended.

Q: What is the recommended method of collection of saliva samples?

A: The passive drool method is recommended for the collection of saliva samples. The passive drool method requires subjects to salivate into a vial or tube.
It has been confirmed that samples collected using cotton swabs or inert polymer swabs can be used similarly to samples collected by the passive drool method. However, caution should be taken, because some swab materials may absorb target analytes.

Q: What should I do if I could not get a good result with a saliva sample?

A: Thick saliva may not give expected results. In such a case, remove insoluble substances, such as mucin, from the saliva sample by freezing and thawing once, followed by centrifugation (example: 1,500 x g for 15 min), and use the supernatant for the assay.

Q: Which extraction buffer is recommended for the analysis of mouse brain lysate?

A: Use RIPA buffer containing a protease inhibitor. To eliminate the influence of surfactant components in the buffer, dilute it with the buffer included in the kit, by a factor of at least twice.

Q: What is the recommended dilution ratio for each sample?

A: The recommended dilution ratio is as follows. It is recommended that you optimize the dilution ratio to fall within the range of standard curve concentrations.

Sample	Dilution ratio
Mouse serum and plasma	4-fold
Mouse brain lysate	2-320-fold
Rat serum	50-fold
Rat plasma	30-fold
Human serum	10-2,000-fold
Human plasma	10-80-fold
Human saliva	2-8-fold

Q: What reagents, instruments, and equipment are required for the assay using this kit?

A: The following are required.

• Purified water（distilled water）
• Test tubes for dilution of standard solution and samples
• Glass utensils for dilution of Wash Solution (graduated cylinder, beaker)
• Pipettes with disposable tips (one capable of pipetting 10μL of liquid accurately and one capable of pipetting 200 to 500 μL)
• Repeater pipette, capable of repeatedly dispensing 100μL
• Water-absorbent material such as a paper towel (to remove any solution remaining on the plate after washing)
• Vortex-type mixer
• Microplate shaker (range approx. 500 to 800 rpm)
• Automatic washer for 96-well plate (if available) or washing bottle
• Microplate reader capable of measuring at 450 ± 10 nm, with the correction wavelength set at 600 to 650 nm
• Software for data analysis

Q: Is it possible to purchase the antibodies provided in this kit?

A: These antibodies are not sold separately.

Q: Can I divide plates?

A: Since the plate in this kit are separable and each row (8 wells) is detachable, divided use of the kit is possible.