Atpase activity - urgent (May/01/2011 )

Hi

I am doing ATPase activity test in the presence of different substrate concentrations and I need to use Lineweaver–Burke plot, but I don't have clear protocol which I can follow, can anybody help.

you plot 1/v vs 1/s (v=reaction velocity, s=substrate concentration). see the wikipedia page for a simple explanation.

or do you have a different question, like how to determine the velocity?

my supervisor suggested to measure the ATPase activity by using increasing ATP concentrations by using malachite green, and I don't know how to set up the protocol and how to measure the differences of increase by lineweaver-burk formula and do I need to use spectrophotometer and at what OD?

Many thanks

I am using innovabioscience kit for ATPase.
.

first you need an understanding of enzyme kinetics. see this wikipedia page for a simple start.

what you must do is assay the enzyme with various concentrations of atp. the assay consists of incubating the enzyme with substrate for a period of time, stopping the reaction and performing a colorimetric determination of the released phosphate.

the innova biosciences kit sets this all out and tells you how to do it.

when you have the results of the assay you can plot them in a number of ways: v vs s, 1/v vs 1/s, v/(vmax-v) vs s, etc.

1/v vs 1/s is the lineweaver-burke plot.

can you give me brief explanation of (lineweaver-burke plot) and how to do the blotting , also can you check the below website, do you think it is helpful.


http://cti.itc.virginia.edu/~cmg/Demo/kinetics/lb/lb/lbApplet.html

rimal on Wed May 4 08:46:09 2011 said:

take the reciprocal of the activity for each assay and plot against the reciprocal of the substrate concentration for that data point. then draw a straight line through the data and the axes.

i can't access that website from here, it's blocked by our firewall.