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.
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