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 Eco RI 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.

Bradford Assays

The Bradford assay was made as shown in the following table.

Tube Fraction Salt Volume Of 1X Eq 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

Table 2. Bradford Assay.

Testing Enzyme Activity

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 EcoRI RXN 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

Table 3. Enzyme reaction with DNA.

Spin samples then incubate in water bath at 37C for 20 minutes. Quick spin after
incubation and then 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 2 4 6 8 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 purified 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 last
four fractions were incubated with -DNA on agarose gel to further examine the presence of the
EcoRI enzyme. When observing the results from the agarose gel, it is clear that there was some
error in this part of the experiment. The expected result was the presence of at least two bands
for each protein fraction, as this would indicate that the -DNA has at least site for Eco RI to
cleave. This could be due to an error in preparation of the gel, because other members of the
class had a similar result.