336 Maundersfree of such activities as DNA endo- and exonucleases, RNase, Pro-
tease, and adenosine deaminase.
BAP is commonly available at 30-40 U/mg protein (with the pro-
viso mentioned above), and CIAP at up to 900 U/mg protein. The
enzyme is supplied at concentrations of 1-50 U/~tl_,, often in abuffer
containing 3.2M ammonium sulfate (65% saturated), 1 mM MgC12,
and 0.1 mM ZnC12. Desalting to remove the ammonium sulfate if
present is necessary prior to reaction.
It is possible to dilute the enzyme for a short time in reaction buffer
(see Section 4.2.), but for longer term storage, the following specific
buffer is recommended:
- 10 mM Tris-HCl (pH 8.0-8.3)
- 1-5 mM MgCI 2
- 0.1-0.2 mM ZnC12
- 50% Glycerol
Triethanolamine (30 mM, pH 7.6) may be used as an alternative to
Tris-HC1, and optional additions include 50 mM KC1 and 3 mM NaC1.
The diluted enzyme is best stored at 4°C rather than at-20°C. Under
these conditions, it is stable for 6 mo. The enzyme is in fact stable for
several days at room temperature in neutral or mildly alkaline solu-
tion, but is inactivated by acid.
4.2. Reaction Conditions
The following reaction conditions are largely optimized for CIAP,
although most are applicable to BAP also. The major difference is that
reactions with CIAP are best performed at 37°C, whereas BAP is used
at 60-65 °C. CIAP has largely superseded BAP as the enzyme of choice,
since it can be easily inactivated after reaction because of its greater
heat lability.
Alkaline phosphatase is active in a wide range of buffers, and it is
often possible to add CIAP directly to restriction endonuclease buffers
after restriction of the substrate DNA, since the enzyme appears to
work equally well as in its own specific buffer. One can add ZnC12 to
1 mM and then proceed (6) or even include the CIAP in the digestion
mixture to act concurrently with the restriction endonuclease (39).
Where a prepurified DNA substrate is available, the phosphatase
reaction can be performed in the following buffer: 10-50 mM Tris-HC1,