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Chromatography
By Grant Akalonu
CHE 447-01
Objective/Introduction
The purpose of this experiment was to purify a restriction enzyme, EcoRI. The enzyme
was purified using ion exchange chromatography. Ion exchange chromatography purifies
solutions of macromolecules such as proteins based on the sign of the charge of the molecule. As
a Type II restriction endonuclease, it cleaves DNA at fixed positions with respect to their
recognition sequence, creating reproducible fragments and distinct gel electrophoresis patterns.
In this experiment fractions of the purified enzyme will be studied using the Bradford assay, and
its enzymatic activity was examined by incubating it with DNA and using agarose gel
electrophoresis to visualize the test results.
Materials/Methods
The stock buffer was prepared by mixing 350 mL of distilled water with 50 mL of 10X
Buffer and 100 mL of 50% glycerol. Then making three salted buffer following Table 1.
1 mL of EcoRI was pipetted into the column in a circular motion and the displacement
was collected immediately as the first fraction. First, we waited until the sample set inside
matrix, and then added 3 mL of 1X Equilibration Buffer and collected fractions 2 and 3
with 1.5 mL each. Second, we added 3 mL of salted buffer I (0.1 M) and collected
fractions 4 and 5 with 1.5 mL each. Third, we added 3 mL of salted buffer II (0.2 M) and
collected fraction 6 and 7 with 1.5 mL each. Lastly, we added 3 mL of salted buffer III
(0.5 M) and collected fraction 8 and 9 with 1.5 mL each. All fractions were stored on ice.
SDS-PAGE Electrophoresis
BradfordAssays
TheBradfordassaywasmadeasshowninthefollowingtable.
Table2.BradfordAssay.
TestingEnzymeActivity
1 0 40 5 5 50
2 6 20 5 5 50
3 7 20 5 5 50
4 8 20 5 5 50
5 9 20 5 5 50
Table3.EnzymereactionwithDNA.
Spin samples then incubate in water bath at 370C for 20 minutes. Quick spin after
incubation before add 5 L of 10x loading gel solution to each sample.
Load 25 L of each sample into the gel and put to run at 135V for almost 1 hour. Then
stained with ethidium bromide for 10 minutes for visualization using a transilluminator.
Fraction Absorbance
Results 1 0.197
2 0.397
EcoRI Bradford Assay
3 3 1.167
2.5
4 0.642
2
Absorbance
1.5 5 0.282
1
6 1.521
0.5
0 7 1.497
0 1 2 3 4 5 6 7 8 9 10
Fraction 8 2.762
9 2.578
1 Molecular
Marker
2 DNA
3 Fraction 6
4 Fraction 7
5 Fraction 8
6 Fraction 9
Based on the results, it appears that the last four fractions of purifies EcoRI solution had
the highest absorbance in the Bradford assay. This is a sensible result, considering that these
fractions were made with the highest concentration of salt buffer in the purification step. The
ions in the salted buffer would compete more with EcoRI to bind with the resin. The expected
result for the agarose gel was
In ion-exchange chromatography, the more ions in the salted buffer, the more it would
compete with the endonuclease enzyme for the binding with resin. As a result, the last four
fractions gave higher absorbance in Bradford assay. In agreement with the Bradford assay, the
SDS-PAGE showed darker bands in the last four lanes that indicates higher concentration of
protein in those fractions. In order to determine if those are the enzyme Eco RI, the fractions
were incubated with -DNA. The result from agarose gel showed that all four fractions - 6, 7, 8,
and 9 - were able to cleave the -DNA. There were two bands for each fraction, so it seems that
there was only one Eco RI site in -DNA.