Overview:
An alkaline phosphatase buffer is required for the detection of alkaline phosphatase activity. It contains 5 – 50 mM MgCl2, 100 mM NaCl and 100 mM Tris.Cl, pH 9.5. In some of the recipes, 0.1 – 1% Tween-20 is also included.
A 100 ml of Alkaline phosphatase buffer of the composition 0.1% Tween-20, 5 mM MgCl2, 100 mM NaCl and 100 mM Tris.Cl (pH 9.5) can be prepared by mixing 0.5 ml of 1 M MgCl2, 2 ml of 5M NaCl, 10 ml of 1 M Tris.Cl (pH 9.5), 0.5 ml of 20% Tween 20 and 87 ml deionized water. You can autoclave the solution and store it at room temperature or in the refrigerator.
REQUIREMENTS
Reagents and solutions
1 M MgCl2
5M Sodium Chloride (NaCl)
1 M Tris-Cl (pH 9.5)
Tween-20
Deionized / Milli-Q water
Equipment and disposables
Measuring cylinder
Conical flask / Beake
Composition:
0.1% Tween-20
5 mM MgCl2
100 mM NaCl
100 mM Tris.Cl, pH 9.5
Note: Tween-20 concentration, ranging from 0.1% to 1%, can be used. Higher concentration helps to decrease precipitate formation.
Objective: Preparation of 100 ml of alkaline phosphatase buffer.
PROCEDURE
Use appropriate personal protective equipment (lab coat, gloves, goggles, etc) for your safety and follow the guidelines of your institute.
Step 1: Take 87 ml Deionized / Milli-Q water in a measuring cylinder. Add the 2 ml of 5M NaCl, 10 ml of 1 M Tris.Cl (pH 9.5), 0.5 ml of 1 M MgCl2 and 0.5 ml of 20% Tween 20. Mix it and transfer to an autoclavable bottle.
Precaution:
Never add all the concentrated stock solutions together and then add water. This can cause the precipitation of solutes. Whenever you are preparing a solution from stock solutions, take the required amount of water in a beaker/measuring cylinder and then add the concentrated stock solution to it.
Step 2: Sterilize the solution by autoclaving (20 minutes at 15 lb/sq.in. (psi) from 121-124°C on liquid cycle).
Tips:
◊ Sterilization is not required if you have prepared it with sterilized stock solutions and water.
◊ One can sterilize the solution by filtering through a 0.2 μm filter unit. Filter sterilization removes all suspended particles with a size of more than 0.2 μm which includes most bacteria and their spores but not mycoplasma. In addition, it does not inactivate enzymatic activities (e.g., DNases). Autoclaving inactivates most enzymes except some (e.g., RNases) and kills most microorganisms including mycoplasma.
Storage:
Alkaline phosphatase buffer can be stored at room temperature or 4°C.
