This protocol is a miniaturization of PAHBAH
assay protocol from Megazyme.
Reagent A: 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.
Reagent B: 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.
Working Reagent: Immediately before
assay, mix Reagents A and B in 1:9 volume ratio. Store working
reagent on ice.
(
1)
Pipet 25 ul of
sample to a
microplate well.
(
2)
Pipet 125 ul of PAHBAH
reagent to
sample well.
(
3) 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.
(
4)
Incubate plate on ice for 5 min.
(
5)
Incubate plate at room
temperature until
plate at rt.
(
6) 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.
Some
microplate 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.