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Methods:CAT assay

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Contents

Purpose

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Assay the activity of the CAT (chloramphenicol acetyl transferase). There are two basic kinds of assays: TLC based and extraction based. In the TLC-based assays, the migration of chloramphenicol or an analog is altered by acetylation. This can be visualized using radioactive chloramphenicol or a fluorescently modified form of the antibiotic (e.g. FastCat). The extraction based assays follow 14C acetate from Acteyl-CoA. AcCoA is mostly water soluble, while Acetyl-Chloramphenicol is extractable into an organic phase such as toluene. By following the partition of label in the aqueous and organic phases, one can measure product formation in the enzyme assay.

TLC assay

Materials Needed

Protocol

Extraction assay

Materials Needed

Protocol

Links

User notes

See also

Invitrogen's FastCat substrate (pdf)

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