Product Uses Include
There are a multitude of enzymes that hydrolyze ATP or GTP to form ADP or GDP and inorganic phosphate (Pi). The Enzyme Linked Inorganic Phosphate Assay (ELIPA) from Cytoskeleton Inc. allows one to measure the phosphate released during hydrolysis on a real time basis. Thus a kinetic assay technique is produced for your enzyme’s activity. Particular purified enzymes that are applicable to this analysis are signaling phosphatases, apyrase, kinesin motors (see Fig. 1), metabolic enzymes, membrane transporters and alkaline phosphatase. Generally this assay is useful for enzymes with Kcat above 0.1, if your enzyme or the family of enzymes has a lower Kcat then the CytoPhosTM Phosphate Assay (Cat. # BK054) is useful. If your enzyme has a very low activity i.e. small G-proteins, the non-radioactive GAP assay (Cat. # BK105) or the radioactive alternative, EasyRad Phosphate Assay (Cat. # BK055) is ideal.
The assay is an adaptation of a method originally described by Webb for the measurement of glycerol kinase plus D-glyceraldehyde ATPase activity and for actin activated myosin ATPase (1). The assay is based upon an absorbance shift (330-360 nm) that occurs when 2-amino-6-mercapto-7-methylpurine ribonucleoside (MESG) is catalytically converted to 2-amino-6-mercapto-7-methyl purine in the presence of inorganic phosphate (Pi). The reaction is catalyzed by purine nucleoside phosphorylase (PNP). One molecule of inorganic phosphate will yield one molecule of 2-amino-6-mercapto-7-methyl purine in an essentially irreversible reaction (2). Thus, the absorbance at 360 nm is directly proportional to the amount of Pi generated in the reaction.
The kit contains sufficient material for 96 assays. THe following components are included:
Todd et al., 2012. Detailed kinetic analysis of the φ29 DNA packaging motor providing evidence for coordinated intersubunit ATPase activity of gp16. Virology. 432, 370-375.
Rajapurohitam et al., 2012. Leptin-induced cardiomyocyte hypertrophy reveals both calcium-dependent and calcium-independent/RhoA-dependent calcineurin activation and NFAT nuclear translocation. Cell. Signal. 24, 2283-2290.
Funk et al., 2004. Development of high-throughput screens for discovery of kinesin adenosine triphosphatase modulators. Anal. Biochem. 329, 68-76.
Question 1: What are the recommendations for machine and filter settings for the ATPase/GTPase ELIPA Biochem Kit (Cat. # BK051/052)?
Answer 1: The assay is based upon an absorption shift from 340 nm to 360 nm. It is therefore very important to use a spectrophotometer that has a narrow bandwidth in order that the wavelength for reading the assay does not encroach upon the 340 nm range. It is recommended that a monochromatic spectrophotometer such as a SpectraMax 250 (Molecular Devices Inc.) be used when possible as the bandwidth in these machines is very narrow (2-5 nm). If a filter based system is being used, then it is important to make sure that the filter is 370 nm with a bandwidth of 10 nm or less. With a broad bandwidth filter, you may experience significant background noise and greatly reduced sensitivity. The spectrophotometer should be at room temperature or 37ºC depending on your enzyme and set on kinetic mode. It is recommended to take a reading once every 30 s. The control is a reaction minus enzyme which will serve as a background control. Do not pre-read the microtiter plate. Start the kinetic readings at time zero. Make sure that all equipment and pipettes are clean and free of inorganic phosphate (Pi), as this assay measures Pi generation.
Question 2: What are the recommendations for my test protein buffer when using the ATPase/GTPase ELIPA Biochem Kit (Cat. # BK051/052)?
Answer 2: We recommend that the test protein be at a concentration of 1 mg/ml and >90% purity. If the test protein exists in a less pure state, we recommend increasing the total protein concentration to compensate for the lower purity. The protein must be in a phosphate free buffer such as Tris-HCl or Hepes (PBS is not suitable). The test protein may also require a cofactor. The protein cofactor or buffer condition (e.g., microtubules for motor proteins, sometimes an alkaline pH buffer, or a certain ion such as calcium for FtsZ proteins) must also be free of phosphate ions. A good starting buffer is 20 mM Hepes pH 7.4 plus 20 mM KCl, plus protease inhibitors, phosphatase inhibitors and any co-factors that are necessary.