Phosphate Quantification Biochem Kits

Cytoskeleton Inc. has introduced the largest range of highly robust phosphate assays currently available.

 

Hydrolysis of… Activity Read out Cat nr Name How it's work
Ubiquitous (ATP, GTP & others), in solution
 
 In high protein concentration solutions (>0.5 mg/ml) End point or kinetic  BK050  PhosFree phosphate assay  Measure liberated phosphate via binding to a reporter dye or by enzymatic conversion into reporter molecule
 In low protein concentration solutions (<0.5 mg/ml). Kcat 0,01 to >1,0 End point assay  BK054  CytoPhos phosphate assay  Measure liberated phosphate via binding to a reporter dye or by enzymatic conversion into reporter molecule
 ATP  Kcat 0,05 to >1,0 Kinetic assay  BK051  ATPase ELIPA kits  Measure liberated phosphate via binding to a reporter dye or by enzymatic conversion into reporter molecule
 ATP & GTP  Kcat 0,0001 to 0,01 Endpoit & Kinetic assay  BK055  EasyRad phosphate assay  Measure liberated radioactive phosphate
 GTP  Kcat 0,05 to >1,0 Kinetic assay  BK052  GTPase ELIPA kits  Measure liberated phosphate via binding to a reporter dye or by enzymatic conversion into reporter molecule
 Small GTPase  - End point assay  BK105 RhoGAP assay Biochem Kit  This GAP assay kit measures the amount of inorganic phosphate (Pi) that is produced as a result of G-protein dependent hydrolysis of GTP to GDP + Pi.
Microtubule induced kinesin ATPase or F-actin induced myosin ATPase  -
 
End point assay BK053 Kinesin HTS ATPase end point assay Measure liberated phosphate via binding to a reporter dye or by enzymatic conversion into reporter molecule
Kinetic assay BK060 Kinesin ELIPA Biochem Kit Measure liberated phosphate via binding to a reporter dye or by enzymatic conversion into reporter molecule

BK051, BK052, BK053, BK054 and BK055 are all suitable for HTS applications and our experienced technical assistance can help you incorporate these assays into your HTS facility.

ATPases and GTPases liberate free phosphate (Pi) from their respective triphosphate nucleotide. Biochem Kits™ BK050, BK051, BK052, BK053 and BK054 measure liberated phosphate via binding to a reporter dye or by enzymatic conversion into a reporter molecule, whereas BK055 uses a radioactive γ-phosphate labeled NTP (or other radioactive phosphate labeled substrate) to measure liberated radioactive phosphate.

BK053 and BK054 (1 µM Pi detection limit) are suitable for higher activity assays such as microtubule induced kinesin ATPase or F-actin induced myosin ATPase.

BK055 (10 nM Pi detection limit) was specifically introduced for low activity enzymes (Kcat <0.01), which usually turn-over their substrate only once or very slowly. For example the GTPase RhoA hydrolyzes GTP in vitro without effectively releasing the GDP product, therefore the enzyme turns-over its substrate only once and requires a highly sensitive assay for Pi measurement. Similarly polymerized tubulin hydrolyzes the majority of GTP at the ends of microtubules, which reduces the apparent GTPase activity to a low specific activity. Polymerized actin has a similar low apparent ATPase activity, which can only be measured by using the BK055 format.

For specifically detecting the GTP hydrolysis by G-proteins, we have also developed BK105.

BK051, BK052 and BK060 (7 µM Pi detection limit) are suitable for Vmax or Kcat determinations, which require kinetic measurements of activity. These kits require a mid to high level activity ATPase or GTPase for sufficient sensitivity.

For more information contact technical assistance on-line or your local office.
 

 

 

 


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