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Ion Exchange

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.

Salted Buffer 1X KCl (g) Concentration of


EquibrationBuffer Salted Buffer
(mL)
1 100 0.75 0.1
2 100 1.5 0.2
3 100 3.75 0.5

The gel was made by mixing enough DEAE-Cellulose (ion-exchanger matrix B)


thoroughly in 35 mL of 10x equilibration buffer (C) by swirling and let the gel swell in
30 minutes. Then the packed column was washed with 5 mL of 1X Equilibration buffer.

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

Dilute the 10x Tris-Glycine-SDS electrophoresis buffer to 1x Tris-Glycine-SDS


electrophoresis buffer.
Take 20 L of each fraction put in each corresponding labeled centrifuge tubes add 5 L
0
sample buffer to each tube. Heat the sample for 5 minutes at 95 C, quick spin the sample
after heat then load.
Set up the SDS PAGE gel in the running chamber and load samples as in figure 3.
Run the gel at 180V for around 40 minutes then remove it from the plastic case stain
over night with Coomassie stain. Then destain it twice before visualization.

BradfordAssays

TheBradfordassaywasmadeasshowninthefollowingtable.

Tube Fraction Salt VolumeOf 1XEq Bradford Total


Content the Buffer Reagent
Fraction
(L) (L)
(L) (L)

1 1 0 50 750 200 1000

2 2 0 50 750 200 1000

3 3 0 50 750 200 1000

4 4 0.1M 50 750 200 1000

5 5 0.1M 50 750 200 1000

6 6 0.2M 50 750 200 1000

7 7 0.2M 50 750 200 1000

8 8 0.5M 50 750 200 1000

9 9 0.5M 50 750 200 1000

10 Blank 0 50 800 200 1000

Table2.BradfordAssay.
TestingEnzymeActivity

Dilute the 50x TAE electrophoresis buffer to 1x TAE electrophoresis buffer.


Make one 0.8% agarose gel by mixing 0.23 g of UltraSpec-Agarose powder in 30 mL
1x TAE electrophoresis buffer. Heat the mixture using microwave oven to dissolve the
agarose powder. Cool the agarose solution to 60C before pouring it into the bed with the
comb sitting firmly and evenly across the bed.
Using the result from Bradford assay to determine which fractions will be used to test the
enzyme activity.
The samples were made according to table 3 and also in the order from left to right.

Tube Fraction Qualified EcoRIRXN DNA Total


Water
Buffer (L) (L)
(L)
(L)

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

Fig. 1. EcoRI Bradford Assay


Table 4. Bradford Assay
Lane Sample

1 Molecular
Marker

2 DNA

3 Fraction 6

4 Fraction 7

5 Fraction 8

6 Fraction 9

Figure 2. Agarose Gel Result for DNA.

Discussion & Conclusion

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.