LacZ activity was assayed using a colorimetric assay based on the reaction of LacZ with ONPG (ortho-nitrophenyl--galactoside), with product concentration measured at 420 nm. To allow calculation of specific activities, the amount of protein in samples was determined by the method of Lowry (J. Biol. Chem. 1951, 193: 265-275).
Urgent help please, thanks.
I don't understand your question, since you have already all the results to calculate the specific activity.
But usually lot of labs uses the Miller technics to measure the specific B-gal activity (Miller, J. (1992) Experiments in Molecular Genetics. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.).
Be careful of your growth state to measure the promoter strength (i. e usually in exponential phase)
The actual question is:
1. Measurement of LacZ activity.
LacZ activity was assayed using a colorimetric assay based on the reaction of LacZ with ONPG (ortho-nitrophenyl--galactoside), with product concentration measured at 420 nm. To allow calculation of specific activities, the amount of protein in samples was determined by the method of Lowry (J. Biol. Chem. 1951, 193: 265-275).
Devise a protocol to allow the specific activity of LacZ in a sample to be deduced. The sample is a 10 ml culture of Escherichia coli.
I need this
I still don’t understand what protocol have you used for measurement of your LacZ activity? Have you extracted the LacZ protein from the cells? Have you noticed the time of the reaction etc… It’s still not clear for me...
If you have used something like Z buffer and chlorophorm, it’s the Miller protocol. So if you have also measured the OD at 550 for protein correction, your can use the Miller's formula.
Miller Units = 1000 x [(OD420 - 1.75 x OD550)] / (T x V x OD600)
With:
OD420 and OD550 from the reaction mixture.
OD600 reflects cell density in the washed cell suspension.
T = time of the reaction in minutes.
V = volume of culture used in the assay in mLs.
The Millers units give the change in A420/min/mL of cells (estimated by OD600)
So alternatively, I think you can change the number of cells by your quantity of protein to have results in A420/min/ml/quantity of protein...
I still haven't used a protocol. The question is asking me to devise a protocol myself for measuring LacZ activity from a 10ml E. Coli sample.
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