Assignment 8 - b-Galactosidase Assay
This is mock data for your current project.
Protein was extracted from IPTG induced cells and assayed for total protein. The extract was poured over a Ni-NTI column and washed with buffer. The 6His-recombinant protein was eluted with elution buffer and assayed for total protein.
Duplicate samples from the crude extract, the flow-through, the washes and the eluted 6His-recombinant protein fractions were assayed for the presence of b-galactosidase activity and compared to a dilution curve generated from pure b-galactosidase. The results are shown below:
The absorbance at 600 nm for the 1 ml cell culture was 0.2 and the conversion of absorbance to cells/ml is: 1A600 = 8*10^8 cells/ml. The crude extract was made from 5 ml of cells.
|Sample||Crude extract||Column flow-through||Column wash||Column elution||Standard
|Total volume (ml)||10||10||40||1||0.001|
|Absorbance 415 nm||1/1 dil, 15µl 1.5, 1.5||1/1 dil, 15µl 0.108, 0.110||1/1 dil, 15µl 0.084, 0.085||1/1 dil, 15µl 1.5, 1.5||no enzyme
|1/2 dil, 15µl 1.5, 1.5||1/2 dil, 15µl 1.5, 1.5||1/1 dilution, 1 unit
|1/4 dil, 15µl 1.5, 1.5||1/4 dil, 15µl 1.5, 1.5||1/2 dilution, 0.5 units
|1/8 dil, 15µl 0.177, 0.175||1/8 dil, 15µl 0.886, 0.890||1/4 dilution,
Note that 15 µl of crude or column extract was used in these assays.
One unit of purified enzyme was used in the 1/1 dilution for the standard curve.
The background is the assay with no enzyme.
Calculate the enzyme units for each dilution in the standard curve. Subtract the background average from each of the average A415 values to get the response due to the enzyme. Plot the standard curve with the enzyme units on the X-axis and the enzyme response absorbance at 415 nm on the Y-axis. From the curve, or appropriate regression, estimate how many units of enzyme are found in the crude and column samples. Calculate how many total units are contained in each of these samples. Calculate how many total mgs of protein are contained in each sample. The specific activity of an enzyme is defined as the number of units of enzyme present per mg of protein. It is an indicator of the purity of the enzyme and the higher the value the more pure the enzyme. Calculate the specific activity of the crude and column samples by dividing the total number of units in each sample by the total mgs of protein in each sample.
Show the results in a table:
|Sample||Total Protein (mg)||Total Units||Specific Activity (U/mg)|
What is the % yield of your protein purification (total units of purified enzyme / total units in crude extract x 100)?
How much have you been able to purify the enzyme taken from the crude extract (fold purification = SA of purified enzyme / SA of crude extract)?
On the average, how many units of enzyme are found in each E. coli cell?
Is the specific activity of the column eluate the same as the specific activity of the purified b-galactosidase used in generation of the standard curve? If not, then offer an explanation of why it is not the same (don't blame assay error since the replication error is very low).
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