Which of the PiColorLock™ family of reagents will be best suited to my assay conditions?
The ALS (which has replaced PiColorLock P) and Gold formulations are both compatible with acid-sensitive and acid-stable phosphorylated substrates, thus the nature of the substrate not significant. A special stabilizer prevents high backgrounds normally caused by non-enzymatic hydrolysis of substrates in highly acidic medium.

The principal difference between the two reagents is the ratio of sample to reagent. In the case of the ALS reagent, the ratio (sample:reagent) is 1:4, whereas for the Gold reagent the ratio is 4:1. Thus if you are running assays in 96-well plates you will not be able to use ALS formulation if your assay volume is >50ul. The assay volume may be as high as 200ul if you use PiColorlock Gold.

If you are developing a new microplate assay, choose an assay volume that gives a depth of liquid (after addition of PiColorlock reagent) that is ~80% of the maximum possible for your particular type of plate. Since absorbance is proportional to path length (depth of liquid), this approach ensures highest possible sensitivity. Thus, an assay with a final volume (including Colorlock reagent) of 100ul might be better run in 384-well plates than 96-well plates.

Regardless of plate type, assays carried out with Gold reagent will always be 4-5 times more sensitive than those carried out with ALS, other considerations being equal, primarily because of the different sample:reagent ratios.

ALS is best for assays carried out in small volumes (relative to total well volume) and where the level of Pi production is in the range 20-200uM (OD ~1 with 100uM Pi). Gold reagent is compatible with almost assay volume and allows much more sensitive measurement of Pi (range 4-40uM; OD ~1 with ~20uM Pi).

Do I have to read the assays quickly?
No. Although PiColorlock is based on malachite green dye, which is normally extremely prone to precipitation, our formulation has special additives and it is not necessary to count quickly. PiColorlock does give rapid colour development if you are in a hurry, but the dye-Pi complexes are also stable for hours, which is useful if there is a queue on your plate reader.

How stable are the reagents?
PiColorLock™ reagents are stable for at least 12 months at +4°C.