PURIFICATION OF PROTEINS

I. Introduction

Gel filtration chromatography is a type of partition chromatography used to separate

molecules in terms of molecular sizes. The principle of gel-filtration is relatively simple.
Molecules are partitioned between a mobile phase and a stationary phase comprising a porous
matrix (of defined porosity) as a function of their relative sizes. This technique is also referred to
as gel-permeation, size-exclusion, and molecular sieve chromatography (Fagain et al., 2009).

The basic components of a gel filtration experiment are the matrix, chromatography
column, and elution buffer. The matrix is the material located in the column which serves as the
stationary phase of the chromatography (The Biotechnology Education Company, n.d.).
Commonly used gel-filtration matrices consist of porous beads composed of cross-linked
polyacrylamide, agarose, dextran, or combinations of these, and are supplied either in suspended
form or as dried powders. The matrix should be compatible with the properties of the molecules
being separated and its stability to organic solvents, pH and temperature should also be
considered (Belewa at al., 1978).

A column containing of such a matrix, typically in bead form, will have two measurable
liquid volumes, the external volume, consisting of the liquid between the beads, and the internal
volume, consisting of the liquid within the beads. The external volume is referred to as the void
volume (V0), while the sum of the external and internal volumes is the total volume (Vt) (Fagain
et al., 2009).

Following sample application, molecules larger than the pores of the stationary phase
matrix will be excluded from the internal volume within the beads. They will, therefore, migrate
quite rapidly through the column, emerging at V0, while molecules smaller than the matrix pores
(as well as those intermediate in size) will equilibrate with both the external and internal liquid
volumes, causing them to migrate much more slowly and emerge at a volume (Ve) greater than
V0. Molecules are, therefore, eluted in order of decreasing molecular size. The elution volume,
Ve, of a particular molecule depends on the fraction of the stationary phase available to it for

Experiment 5: Purification of Proteins 1

diffusion. This can be represented by the constant Kd or Kav (also referred to as the partition
coefficient) (Fagain et al., 2009). Therefore:

Ve = V0 + Kav (Vt - V0)

Rearranging this equation gives:

Kav = (Ve - V0)/(Vt - V0)

Lastly, the elution buffer is the mobile phase of the chromatography which flows through
the matrix and out of the column. The column, with the matrix and sample, is developed by the
elution buffer. This means that the molecules in the sample are carried by the flow of buffer into
the matrix where they are gradually separated. The separated zones of molecules then flow out of
the column where they are collected for analysis (The Biotechnology Education Company, n.d.).

Factors such as matrix choice, sample size and concentration, column parameters, choice
of eluent, effect of flow rate, and column cleaning and storage, should be considered when
designing a gel-filtration system (Fagain et al., 2009).

One of the main advantages of gel filtration chromatography is that separation can be
performed under conditions specifically designed to maintain the stability and activity of the
molecule of interest without compromising resolution. This separation technique, however, also
has disadvantages. Because of the large size of gel filtration columns, large volumes of eluent are
usually required for their operation, often creating excessive running costs. It also has an inherent
low resolution compared to other chromatographic techniques, because none of the molecules
are retained by the column and non-ideal flow occurs around the beads. Despite these
disadvantages, gel filtration chromatography still holds a key position in the field of biomolecule
separation because of its simplicity, reliability, versatility and ease of scale-up (Fagain et al.,
2009).

Gel filtration chromatography, with its unique mode of separation, has been used
successfully in the purification of thousands of proteins and peptides from various sources. In
this experiment, egg albumin was purified by the use of this technique.

Experiment 5: Purification of Proteins 2

 II. Methodology

III. Results and Discussion

Protein purification is essential in the characterization of the function, structure, and
interactions of proteins. It involves a series of steps which includes cell lysis, separation of
soluble protein components from cell debris, and separation of protein of interest from product
and process - related impurities. The isolation and purification of proteins is an important aspect
of modern biochemistry as it allows scientist to study and understand a particular proteins
function.
The experiment focused on the purification of albumin sample. A pre swollen gel, made
from Sephadex G-100, was used as molecular sieving matrix which separate molecules of
different sizes. It consists of cross-liked polymers that are generally inert, do not bind or react
with the material being analyzed, and are uncharged. However, it cannot be used to separate
biopolymers larger than 300,000 Daltons. Other types of sieving matrix that may be used for gel
filtration chromatography are agarose and polyacrylamide. Agarose gels, due to their greater
porosity, may be used to separate molecules and participates up to a molecular weight of several
million Daltons. Polyacrylamide gels, on the other hand, can be used to separate molecules of up
to 300,000 Daltons.
Gel filtration chromatography column bed has three functional components: the pore
volume, the void volume, and the matrix volume (bed volume). The pore volume refers to the
pore lumen space within the particles. The void volume refers to the excluded volume i.e., the
space between the particles. And the matrix volume refers to the solid component of the particles
that fills the column bed.

Experiment 5: Purification of Proteins 3

 1.4
1.2
1

Absorbance 0.8 Absorbance @ 370

0.6 Absorbance @ 500
0.4
0.2
0
-10 10 30 50 70 90 110 130
Elution Volume (mL)

Figure 1. The column calibration curve.

Figure 1 presents the absorbance of different fractions plotted against the eluent volume
of the calibration standards. The column was first calibrated in order to measure the void volume
of the column with the aid of blue dextran. In the case of blue dextran, the molecules are colored
blue which helps in checking visually the elution from the column easily. Gel column profiles
are summarized in Table 1.
Table 1. The column profile.
Internal diameter (cm) 2.5
Height of gel bed (cm) 6
Column volume, (mL) 29.45
Void volume (Vo), mL 5.04
Total volume (VT), mL 29.5
Allowable amount of sample 1 5% of VT

The pore diameter defines the exclusion limit of the gel. Proteins that are too large to
enter the pores are excluded and have access to only the void volume. Proteins larger than the
exclusion limit elute together in a single peak at the beginning of the chromatography - the void
volume. Proteins molecules that are smaller than the pore size enter the particles and their
separation is determined by the pore size distribution within the pore volume. Among these
proteins, the larger proteins are eluted earlier than the smaller protein molecules giving rise to the
fractionation of protein molecules based on their size. Hence, the larger the beads, the more rapid
the flow rate and the poorer the resolution. This is because as the flow rate increases, the time

Experiment 5: Purification of Proteins 4

available for the molecules to equilibrate between the mobile phase and the pore space in the
stationary phase decreases. The larger beads are for very large preparation in which resolution is
less important than time. While super fine is used if maximum resolution is required for example
for analytical work but it is very slow.
0.16
0.14
0.12
Absorbance

0.1
0.08
A370
0.06
A500
0.04
0.02
-2.78E-17
0 20 40 60
Elution volume (mL)

Figure 2. The elution volume of sample vs. absorbance.

Figure 1 presents the absorbance of different fractions plotted against the eluent volume
of the sample.

1.4

1.2

1
Absorbance

STD_A370
0.8
STD_A500
0.6
Sample_A370
0.4 Sample_A500
0.2

0
0 20 40 60 80 100 120
Elution volume (mL)

Figure 3. The elution volume of standard and sample vs. their corresponding absorbance.
The advantage of gel filtration for the separation of molecules differing in molecular
weight is that it doesnt depend on temperature, pH, ionic strength and buffer composition.
Therefore, separation can be carried out under any conditions. There is less adsorption and less

Experiment 5: Purification of Proteins 5

zonal spreading than in other techniques. Moreover, the elution volume is also related to the
molecular weight.
Another technique which can be used in protein purification is affinity chromatography.
In this technique the stationary phase consists of a support medium (cellulose beads) on which
the substrate (or a coenzyme) has been bound covalently in a way that the reactive groups
essential for enzyme binding are exposed. As the mixture of proteins is passed through the
chromatography column, those proteins that have a binding site for the immobilized substrate
will bind to the stationary phase, while all other proteins will be eluted in the void volume of the
column. Once the other proteins have all been eluted, the bound enzymes can be eluted in
various ways: by increasing the ionic strength of the buffer, by changing the pH of the buffer,
and by adding a high concentration of substrate (or a substrate analogue) to the elution buffer.
Gel filtration, on the other hand, allows polypeptide to be purified from other different sized
polypeptide by passing through a gel filtration medium packed into the column. Unlike affinity
chromatography, fractions passing through the column dont bind to the medium.

IV. Conclusion

V. References
Fagain, C., Cummins, P., OConnor, B., Gel Filtration Chromatography
http://doras.dcu.ie/17808/1/Gel_Filtration_Chapter%2711.pdf (accessed Oct 20, 2017).
The Biotechnology Education Company, Principles of Gel Filtration Chromatography
https://www.conatex.com/media/manuals/BAEN/BAEN_1093147.pdf (accessed Oct 20,
2017).
Belewa, M., Poratha, J., Fohlmana, J. and Janson, J.-C. (1978) Adsorption phenomena on
sephacryl S-200 superfine. J. Chromatog. A 147, 205-212.
n.d. What is Protein Purification? Retrieved October 24, 2017 from
https://www.thermofisher.com/ng/en/home/references/protein-analysis-guide/affinity-
chromatography/what-is-protein-purification.html.

Experiment 5: Purification of Proteins 6

 Alzahrani, Z. (2010). Gel Filtration Chromatography. KSU - College of Science -
Department of Biochemistry
Fish, W. W., Mann, K. G., and Tanford, C. (1969). The Estimation of Polypeptide Chain
Molecular Weights by Gel Filtration in 6 M Guanidine Hydrochloride. THE JOURNAL
OF BIOLOGICAL Chemistry. Vol. 244, No. 18, Issue of September 25, pp. 4989-4994

VI. Appendices

Appendix A. Tables
Table 2. Parameters in gel chromatography.
Sample volume (mL) 1
Flow rate (mL/min) 2.26

Table 3. Absorbance of different fractions collected after gel chromatography of the sample.
Fraction Volume
Test tube number Ve (mL) A370 A500
(mL)
1 11.94033 11.94033 0.009 0.009
2 7.307333 19.24767 0.158 0.079
3 7.6388 26.88647 0.035 0.019
4 6.516333 33.4028 0.022 0.014
5 5.872233 39.27503 0.012 0.013
6 6.098233 45.37327 0.012 0.009
7 7.4806 52.85387 0.009 0.008
8 8.693467 61.54733 0.009 0.005
9 6.866633 68.41397 0.008 0.009

Table 5. Absorbance of different fractions collected after gel chromatography of the calibration
standards.
Test tube Fraction Volume (mL) Ve (mL) A370 A500

Experiment 5: Purification of Proteins 7

 number
1 5.0424367 5.042436667 0.009 0.01
2 5.9464367 10.98887333 0.005 0.011
3 2.1688467 13.15772 0.04 0.035
4 2.98772 16.14544 0.271 0.22
5 4.73922 20.88466 0.061 0.029
6 2.1364533 23.02111333 0.049 0.04
7 1.91196 24.93307333 0.06 0.08
8 4.4405233 29.37359667 0.66 0.55
9 5.6677033 35.0413 1.5 1.35
10 2.7308333 37.77213333 0.539 0.069
11 2.72669 40.49882333 0.88 0.14
12 2.9248167 43.42364 0.12 0.035
13 4.5147267 47.93836667 0.305 0.242
15 2.97077 50.90913667 0.182 0.058
16 5.8259033 56.73504 0.19 0.081
17 7.2504567 63.98549667 0.089 0.067
18 5.5859667 69.57146333 0.051 0.07
19 6.08053 75.65199333 0.048 0.045
20 6.2982433 81.95023667 0.022 0.03
21 6.53479 88.48502667 0.025 0.02
22 7.1028033 95.58783 0.018 0.01
23 5.93363 101.52146 0.013 0.02
24 6.2656617 107.7871217 0.009 0.011
25 6.2656617 114.0527833 0.009 0.009
26 7.37212 121.4249033 0.009 0.015
27 6.9871667 128.41207 0.009 0.008

Appendix B. Calculations

= (1.25)2 (6) = 29.45 3

Allowable amount (sample) = 1-5% of VT

Range = (29.45 0.01) = 0.295 to (29.45 0.05) = 1.475

Experiment 5: Purification of Proteins 8

 2.26
= = 15.505 min ( ) = 35.0143

Experiment 5: Purification of Proteins 9