October 20th, 2017

Protein Purification Short Report

MacKenzie Olbrys

1. Introduction

Protein purification is vital to performing biochemical assays that specifically test for the
protein of interest and its effects. There are multiple techniques to purify proteins such as gel-
filtration chromatography, affinity chromatography, high performance liquid chromatography
(HPLC), and ion-exchange chromatography. These techniques purify the protein desired
depending on the protein’s characteristics of size, isoelectric point, binding affinity, structure, or
ultraviolet absorbance.1 The technique used in this study is immobilized metal affinity
chromatography using a column with nitrilotriacetic acid (NTA) agarose as the solid phase.
During this chromatography, Nickel(II) is immobilized due to its affinity to the chelating ligand
NTA as shown in Figure 1.2

Figure 1: Structure of NTA agarose Interacting with Ni2+. In the immobilized metal affinity
chromatography column utilized, NTA agarose, due to the oxygen and nitrogen atoms in the structure, has
the ability to noncovalently bind to Ni2+ to stabilize the metal on the solid phase of the column.2

The purpose of this complex is to extract the desired protein which has a His-tag modification.
Due to the nitrogen groups in histidine, histidine binds to the Nickel(II) and stays in the column
while other contaminants in the solution are washed out. In order to extract the His-tag protein
product, the histidine analog imidazole is used. Due to its similar structure to histidine as shown
in Figure 2, imidazole will compete with the histidine residues to bind to the Nickel(II) through a
process called ligand exchange and release the desired modified protein for collection. 3

Figure 2: Structures of Imidazole and Histidine. Imidazole is an analog of histidine which allows elution in
IMAC because the imidazole can also have interactions with the Nickel(II) and compete with the interactions
histidine has.4
 Once purified, the protein must be visualized using gel electrophoresis on an SDS-page.
Sodium dodecyl sulfate (SDS) denatures the protein and must be used to run the protein samples
in order to yield an overall negatively charged linear fragment of the protein.5 This allows the
protein to move in the downwards direction on the SDS-page based on the size of the protein.
Once purified and visualized, the protein concentration must be determined for further use in
testing like in binding affinity studies. The colorimetric assays available to use for protein
concentration determination test concentration based on specific characteristics of the protein,
and each assay has its limitation. For example, using direct UV light and testing for absorbance
at 280 nm will yield a concentration based on the aromatic residues in the protein such as
tyrosine, tryptophan, phenylalanine.6 Contaminants such as nucleic acids must be considered in
this concentration determination because nucleic acids can show absorbance at 280 nm.
Therefore, absorbance at 260 should be measured to calculate protein concentration and
percentage of nucleic acid contamination as in the Warburg-Christian method.7 The Bradford
assay uses a standard curve from known protein concentrations in Coomassie brilliant blue G-
250 reagent to calculate the concentration. The noncovalent electrostatic, hydrophobic, and Van
der Waals protein interaction with the dye reagent yields an absorbance at 595 nm that can be
measured using UV-Vis spectrophotometry.8

2. Tables and Figures

2.1 Figures

Figure 3: Gel Electrophoresis on a 10% SDS-Page of T7 RNA Polymerase After Immobilized Metal Affinity
Chromatography. After running the gel at 175 V, Coomassie blue stain was used to stain the gel, and the image
above was taken. Lanes 1-8 consist of the following consecutively: 200 KDa protein ladder, pre-induction, post-
induction, flow-through, wash, elution fraction one, elution fraction two, and 200 KDa protein ladder. This gel was
run and imaged in winter 2017semester by graduate student Prabuddha Madubashitha.
 Size Analysis of Purified T7 RNA Polymerase on SDS-Page
2.4
Log(molecular weight in KDa)

2.2

2.0

1.8

1.6

1.4

1.2

1.0
0.0 2.0 4.0 6.0 8.0 10.0 12.0
Distance Traveled (cm) y = -0.0983x + 2.2353
R² = 0.9619
Figure 4: Linear Regression Analysis of Size of Purified T7 RNA Polymerase on 10% SDS-Page. The
measurements of distance traveled were done on a printed picture of the gel measuring with a ruler from the middle
of the starting cell to the middle of the protein band that traveled. Using excel, a linear trend line was created with
the equation and R2 value to indicate the relationship between the data and the trend line. The x values represent the
distance the protein band traveled in centimeters. The y values represent the log of the molecular weight in KDa.

BSA Standard Curve with Phosphate Buffer Blank

1.000

0.800
Absorbance at 595 nm

0.600

0.400

0.200

0.000
0 0 0 1 1 1 1
-0.200
Concentration (mg/mL) y = 0.869x + 0.0525
R² = 0.9867
Figure 5: Standard Curve of Bovine Serum Albumin Absorbance at 595 nm with Phosphate Buffer as a Blank.
Using known concentrations of BSA in phosphate buffer and dye reagent, absorbances at 595 nm were measured
and plotted using excel. The linear regression analysis was performed on excel to yield an equation and R2 value to
relate the data to the trend line. The x values represent concentration of the protein in mg/mL, and the y values
represent the absorbances determined at 595 nm. Values at 1.3 mg/mL and 1.5 mg/mL were omitted for the
preparation of the trend line due to the high and low absorbance values not within the trend.

2.2 Tables
 Table 1: Size Analysis of Purified T7 RNA Polymerase on 10% SDS-Page*

*Measurements are from the SDS-page prepared by graduate student Prabuddha

Madubashitha.

Table 2: Absorbance of Dilution Series of Standard BSA

Table 3: Absorbance of T7 RNA Polymerase Samples

 Table 4: Comparison of Methods for Protein Concentration Determination

3. References

1. CHM 6610 Experiment 6 Protocol and Lecture Notes, Fall 2017.

2. (2017) Protino Ni-NTA Agarose, Bioké.
3. Gaberc-Porekar, Vladka, Menart, Viktor (2001) Perspectives of immobilized-metal
affinity chromatography, J. Biochem. Biophys. Methods, 49, 335-360.
4. (2017) Protein Expression and Purification Series, Biotechnology Explorer BIO-RAD.
5. Ninfa, Alexander J., Ballou, David P., Benore, Marilee, (2010) Fundamental Laboratory
Approaches for Biochemisty and Biotechnology, 2nd edition.
6. Pace, Nick C., Vajdos, Felix, Fee, Lanette, Grimsley, Gerald, Gray, Theronica (1995)
How to measure and predict the molar absorption coefficient of a protein, Protein
Science, 4, 2411-2423.
7. Ninfa, Alexander J., Ballou, David P., Benore, Marilee, (2010) Fundamental Laboratory
Approaches for Biochemisty and Biotechnology, 2nd edition.
8. Compton, Steve J., Jones, Clive G. (1985) Mechanism of dye response and interference
in the Bradford protein assay, Analytical Biochemistry, 151, 369-374.

4. Acknowledgements

Professor Dr. Christine Chow, teaching assistants Fidelis Ndombera and Rabiul Islam,
graduate student Prabuddha Madubashitha, and my colleague Alexsandra Cvetkovska all assisted
in the understanding and execution of this experiment.