Enzymes and Other Products for Molecular Biology, FAQ

CIP, Calf Intestinal Alkaline Phosphatase
Q 01:	What are the typical applications?
Q 02:	Why should I choose CIP instead of the corresponding bacterial phosphatase?
Q 03:	What problem could appear if I use excess amount of CIP?
Proteinase K
Q 01:	What are the typical applications?
Q 02:	Is Proteinase K stable?
Q 03:	Have you any hints for the reaction conditions?
Q 04:	How should I store my Proteinase K?
Q 05:	My lyophilized, reconstituted Proteinase K solution has gone through many freezing and thawing cycles. I have noticed some precipitation in the product. Is the product still usefull?

CIP, Calf Intestinal Alkaline Phosphatase

Q 01: What are the typical applications?
CIP (Calf Intestinal Alkaline Phosphatase) catalyzes the hydrolysis of a wide variety of phosphomonoesters. It catalyzes the removal of 5’ phosphate residues from DNA, RNA and ribo- and deoxyribo-nucleoside triphosphates. Primarily it is used to remove 5’ phosphates from DNA fragments to prevent self-ligation and to remove 5’ phosphates from DNA and RNA prior to labelling the 5’ end with ³²P.

CIP can also be used to remove phosphate groups from serine, threonine and tyrosine residues in proteins.

Q 02: Why should I choose CIP instead of the corresponding bacterial phosphatase?
Although the amino acid sequences of alkaline phosphatase from bacteria, like E. coli and mammals are 25-30% conserved, there are some remarkable functional differences between the amino acid sequences of the active sites of the enzymes. These differences make mammalian alkaline phosphatases 20-30 fold more active than the bacterial phosphatases (1,2).

Bacterial Alkaline Phosphatases (BAP) are also far more heat resistant than the mammalian phosphatases. Therefore it is difficult to completely inhibit BAP at the end of the dephosphorylation reactions (3). In cloning procedures phosphatase must be completely removed after dephosphorylation to make sure that subsequent ligations work efficiently.

References:
(1) Brunel, C. and Cathala, G. (1973) Biochim. Biophys. Acta 309, 104-115.
(2) Cathala, G. et al. (1975) J. Biol. Chem. 250, 6046-6053.
(3) Maniatis, T., Fritsch, E.F. and Sambrook, J. (1989) Molecular Cloning, A laboratory Manual, Cold Spring Harbor Laboratory, pp.572.

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Q 03: What problem could appear if I use excess amount of CIP?
To prepare a plasmid for use in cloning experiments, one usually first digests the plasmid with some appropriate restriction enzyme. Then the plasmid should be dephosphorylated with CIP, phenol/chloroform extracted, ethanol precipitated and dissolved into appropriate buffer. After dephosphorylation CIP should be inactivated. If excess amount of CIP is used, CIP may cause co-precipitation of DNA when inactivating CIP by heat treatment. This problem may be avoided by doing phenol/chloroform extraction carefully right after dephosphorylation step. No heat inactivation is then necessary.

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Proteinase K

Q 01: What are the typical applications?
Proteinase K is a serine protease which cleaves peptide bonds adjacent to the carboxylic group of aliphatic and aromatic amino acids. It shows proteolytic activity as well on denatured proteins as on native proteins.

Proteinase K can be used to isolate high molecular weight DNA, plasmid DNA and genomic DNA. Also RNA can be purified free of RNase.

Q 02: Is Proteinase K stable?
Proteinase K has a broad temperature range. It is active up to 65 °C, after which it starts to denature. pH optimum is 8 while the pH range is 4.3-12. Calcium is a stabilizing factor of the enzyme, however soluble calcium is not essential for enzymic activity. This means that EDTA, which is used to inhibit magnesium dependent enzymes, will not inhibit Proteinase K activity. An interesting feature of Proteinase K is that it can be used in the presence of SDS and urea.

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Q 03: Have you any hints for the reaction conditions?
In general the required conditions depend on what are the demands of the material of interest. A typical working concentration for Proteinase K is 50-100 µg/ml. Good reaction temperature is 37-60 °C. The lower is the temperature the longer is the need for digestion time. The reaction can be terminated by adding an Proteinase K inhibitor, such as PMSF or DFP. TCA precipitation or raising the temperature of the reaction over 65 °C for 15-20 minutes will also terminate the reaction.

Q 04: How should I store my Proteinase K?
Liquid form and lyophilized powder are both recommended to store at -20 °C. Proteinase K is stable for at least 1 year as well as at -20 °C as at +4 °C.

Q 05: My lyophilized, reconstituted Proteinase K solution has gone through many freezing and thawing cycles. I have noticed some precipitation in the product. Is the product still usefull?
If the product is not contaminated, in many cases, yes. Try to dissolve the precipitate by heating the product for 30 minutes at 37 °C or at 55 °C. If this doesn’t help, make a new, fresh solution of Proteinase K. Liquid form Proteinase K is supplied in 50 % glycerol. It retains its activity without precipitation problems during freezing-thawing cycles. It is also in a liquid form at -20 °C, so it is ready to use right away.