BHB Assay
From Bridges Lab Protocols
Materials
- Wako Autokit Total Ketone Bodies: Cat#'s 415-73301, 411-73401 and 412-73791. Once R1 and R2 are resuspended, use within 3 weeks. Protocol modified from Wako Kit Protocol
- Serum, prepared according to Measuring Fasting Insulin
- Microtiter plate
- Plate reader, warmed to 37C
Procedure
- Prepare R1 and R2 if not already resuspended
- Pipet 4 uL of sample into each well (For ketogenic diet samples pipet 1 uL).
- Pipet 0, 1, 2, 3, 4, 5, 6, and 7 uL of 40 uM standard into wells. If evaluating ketogenic diet samples, also pipet 1, 2, 3, 4, 5, 6, 7 and 8 uL of 300 uM standard
- Using multichannel pipet add 270 uL of R1 to each well
- Incubate at 37C for 5 minutes
- Add 90 uL of R2 to each well and mix
- Put in plate reader at 37C for 2 minutes
- Measure A405 at 30s intervals. You may need to increase interval time in order to measure all your wells. Measure for at least 3 minutes total. Modify BHB Assay.prt on the plate reader if necessary.
- Calculate Max V according to the software.
- Calculate Ketone Levels by plotting Rate (Max V) versus BHB amount and adjusting by volume used.
