Affinity resins have been widely used for the purification of enzymes and other proteins that bind nucleotides and related molecules. ATP-agarose resin comprises ATP attached to agarose beads via its gamma-phosphate. A long hydrophilic spacer (14-atom) is used to minimize unwanted hydrophobic interactions and to facilitate unhindered interactions with biomolecules. The ligand is coupled through the gamma-phosphate group which means that the resin is resistant to phosphatases found in many crude tissue extracts.
- Quick and easy-to-use
- Extremely stable
- Can be used in column chromatography or batch processing
- Stringently QC tested
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Q1. What is the best buffer to use?
There is no ‘right’ answer here. In the absence of any information on the binding requirements of the protein(s) of interest a good starting point is a buffer containing 20mM Hepes, 100-500mM NaCl (or KCl), 20mM MgCl2 and 1mM DTT, pH 7.5. Alternatively, try experimenting with several buffer conditions using small amounts of resin in 1.5ml tubes. After the wash step, elute with ATP or other competing ligand and analyse the elutedproteins by SDS-PAGE. The purification method can then be scaled up using the preferred buffer conditions.
Q2. Are there any other ways of eluting binding proteins from the ATP Agarose?
Yes. While ATP is the obvious choice, ligands that are structurally related to ATP may be used (e.g. NADH, AMP, adenosine) to elute a specific subset of the ATP-binding proteins. Drugs that are known to bind to ATP-binding proteins might also be used. If preservation of biological activity is not required, aliquots of resin may be boiled with SDS sample buffer prior to gel electrophoresis.