This protocol is a miniaturization of PAHBAH assay
protocol from Megazyme.
: Slurry 1.0 g of p-Hydroxy benzoic acid hydrazide (PAHBAH) in 6.0 ml of ddH2O. To this, add 1 ml of concentrated HCl. Make up the volume to 20 ml with ddH2O.
: Dissolve 2.49 g of trisodium citrate in 50 ml of ddH2O. To this, dissolve 0.22 g of calcium
chloride or 0.292 g of calcium
chloride trihydrate. To this, dissolve 4.0 g sodium hydroxide. Adjust the volume to 200 ml with ddH2O.
Store both reagents at rt.
: Immediately before assay
, mix Reagents A and B in 1:9 volume ratio. Store working reagent
25 ul of sample
to a microplate
125 ul of PAHBAH reagent
) Float the plate
uncovered on near boiling (>90 °C) water
bath for 6 minutes. Note
: Any method that heats the plate
evenly to nearly 100 °C is acceptable.
) Incubate plate
on ice for 5 min.
) Incubate plate
at room temperature
) Read absorbance
at 410 nm. (If microplate reader
has only wavelengths 405 nm and 415 nm available, either one is OK). Note
: I usually read the absorbance
3 times and average. Remember to wipe the plate
dry before putting it into the reader.
You can add shaking/mixing step after adding the PAHBAH reagent
. I use shaker
only right before reading the absorbance
. When adding PAHBAH reagent
, bubbles from the pipette tip
mix the sample
and the reagent
(push the plunger all the way).
I use multichannel pipette
with every step possible to increase reproducibility
readers get contaminated easily because the tray jerks quickly from to/from the reading position and well contents can splash to the optics
. Lowish total volume of 150 was chosen so that there is no danger or contaminating the reader.
I don't use the edge wells for fractions, I pipet
samples only to the inner 60 wells on a 96-well plate
and 25 ul of water
to the edge wells. Similarly, if assaying few samples, I use wells in a middle off the plate
and surround them with mock samples (=water
). All wells with any content are then treated identically from that point on. This prevents any edge effects due to uneven heating.