https://bridgeslab.sph.umich.edu/protocols/index.php?action=history&feed=atom&title=BHB_Assay 2026-05-30T23:47:43Z Revision history for this page on the wiki MediaWiki 1.45.1 https://bridgeslab.sph.umich.edu/protocols/index.php?title=BHB_Assay&diff=1360&oldid=prev 2017-07-20T21:04:55Z

<p>wrote initial page for total ketone assay</p> <p><b>New page</b></p><div>==Materials==<br /> * Wako Autokit Total Ketone Bodies: Cat#&#039;s 415-73301, 411-73401 and 412-73791. Once R1 and R2 are resuspended, use within 3 weeks. Protocol modified from [http://www.wakodiagnostics.com/applications/microplateapp_autokit_total_ketone_bodies_wls_1973_1.pdf Wako Kit Protocol]<br /> * Serum, prepared according to [[Measuring Fasting Insulin]]<br /> * Microtiter plate<br /> * Plate reader, warmed to 37C<br /> <br /> ==Procedure==<br /> # Prepare R1 and R2 if not already resuspended<br /> # Pipet 4 uL of sample into each well (For ketogenic diet samples pipet 1 uL).<br /> # 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<br /> # Using multichannel pipet add 270 uL of R1 to each well<br /> # Incubate at 37C for 5 minutes<br /> # Add 90 uL of R2 to each well and mix<br /> # Put in plate reader at 37C for 2 minutes<br /> # 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.<br /> # Calculate &#039;&#039;&#039;Max V&#039;&#039;&#039; according to the software.<br /> # Calculate Ketone Levels by plotting Rate (Max V) versus BHB amount and adjusting by volume used.</div>

Davebrid