The Chromatography Method: Scientific Experiment

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Table of Contents

Abstract
Materials Provided
Procedure
Results
Discussion
References List

Abstract

In this experiment, a laboratory test was carried out in order to separate plasminogen from horse serum. In this experiment, an activity chromatography method was used to separate plasminogen from horse serum using lysine sepharose. During the separation and purification, the extracted protein was divided into eight portions of which each portion was assayed at 280 nm optical density. From the results, its concluded that bands of high intensity are believed to contain high plasminogen which is evident by the level of absorbance (Silverstein, 1974). These eight portions were run on SDS- PAGE gel electrophoresis to separate isolated protein from further determination in this experiment; methods of analysis reported provide approximate determination of plasminogen.

In molecular biology, one of the major objectives is separation of a particular protein molecule from a complex of mixtures. This involves a number of steps of which each stage is monitored by electrophoresis to gauge its effectiveness. Chromatography is a technique used to separate molecules based on the differences in their chemicals and physical properties (Nieuwenhuizen and Traas, 1989). Chromatography lacks specificity for a single protein of which affinity chromatography is designed to overcome this limitation through exploitation of the distinctive interaction of a molecule with the next molecule which is a complementary binding molecule called ligand (Kline, 1953).This investigation concerns the purification step of plasminogen from horse serum involving lysine sepharose affinity chromatography. Plasminogen is the inactive precursor of plasmin and its essential in blood coagulation. The protein component is divided into several portions, of which each is assayed at 280nm of optical density measurement. These portions then undergo electrophonesis by being run through SDS-PAGE gel. SDS-PAGE separates isolated protein of which can be visualized by coo massive stain that binds to the experimental proteins and hence, the intensity of this resultant bands is used to give plasminogen estimation in a protein. A successful band is used to estimate the amount of plasminogen in a protein. A successful separation process is vital in classifying functions, protein structure and the relationship of plasminogen

Materials Provided

Column: Affi-Gel Blue gel (BioRad)
Protein Mixture: Horse Serum (Fisher Scientific)
Column buffer: 10 Mm Tris-HCl, 150Mm NaCl, Ph 8.0
Elution buffer:10 Mm Tris- HCl,150Mm NaCl, 1%SDS, Ph 8.0
SDS-PAGE: 4-15% Tris-HCl Gradient ready-Gel (BioRad)
Electrophoresis Buffers: Same as used for SDS-PAGE lab

Procedure

The Affi-gel blue column was prepared. The Affi-gel blue is slurry containing the matrix and a buffer with preservatives.
The slurry was poured into the column and equilibrated the column with the column buffer. Then column was secured by clamping to a ring stand. A porous disc in the bottom of the column retains the gel but lets solution and proteins flow through.
A couple of ml of column buffer was put in the column, and then 2ml was transferred to the slurry column. The buffer was let to drip through into a beaker. Two layers begun to form, with the Affi-Gel Blue on the bottom and buffer on top. When the buffer approached the top of the gel layer, a column buffer was added to the column and allowed to flow through the column to be washed (Hatton and Regoeczi , 1975)
The column material was not disturbed. Washing was repeated. Keeping the Affi-gel wet
The bottom of the column was closed. 0.50ml sample of horse serum was prepared by diluting the serum 1:5 with column buffer. 20 ml of SDS-PAGE saved (Grant, 1990).
To apply the sample, the buffer was allowed to drain to within 1-2mm of the top of the Affi-Gel Blue. Close off the column, and then carefully transferred 0.50ml of serum into the column. The flow was then collected (the solution containing protein that does not bind to the column).
When the serum entered the column, the column was refilled with column buffer and washed the column. Collection of the flow was done until a total volume of about 1.5ml was obtained. The rest was discarded. The column buffer was rinsed to make sure all non-bound protein had been removed.
When the final wash drained to about1-2mm of the top of the Affie-Gel Blue, 2ml of the elution buffer was carefully transferred into the column to remove the protein containing the albumin.
The solution that elutes was collected (the bound protein) for SDS-PAGE. Washing the Affie-Gel Blue with about 10 ml of elution buffer was continued and then rinsed with about 10ml of column buffer. The Affie-Gel Blue was returned to be used used as Affie-Gel Blue.
SDS-PAGE was performed onthe original serum sample, then flow-through/wash fraction, and the bound fraction carried out. Load 20mlof each sample, add 20ml reducing sample buffer, mixed well and boiled. Load 20 ml of each sample and 5 ml of the standards onto the gel, electrophoreses, stain and distain carried out.

Results

READ	ABS	nM
0	-1.9740	280.00
1	1.3186	280.00
2	1.4563	280.00
3	1.4683	280.00
4	1.6110	280.00
5	0.4301	280.00
6	0.3674	280.00
7	0.3656	280.00
8	0.3554	280.00

Discussion

Affinity chromatography provides room for fractional plasminogen distribution. In this process plasminogen and several forms of protein type are differentiated basing on the molecular size (Brockway and Castellino, 1971). From the results, it can be shown that the larger sized molecular proteins diffuse at slower rate compared to the smaller ones and hence this cover a shortest distance from origin. In this experiment, the size of the plasminogen has a smaller size of 92kDa (Swain, 1998):.The results reflects several sharp bands when separated by electrophoresis depending on the time limit running time for electrophoresis should be longer hence this is an indication of successful isolation of plasminogen from horse serum (Deutsch and Mertz, 1970)

Generally intensity of band is proportional to the amount of protein present in band which is also proportional to the amount of plasminogen at 280nm tube 3 are highest absorbance and its expected to contain at highest level of plasminogen while tube 8 gave the smallest absorbance, hence contains the smallest amount of plasminogen, the high absorbance rate obtained in the tube 4 may be due to great amount of undesired proteins (Williams et al, 1992)

References List

Brockway, W. J. and F. J. Castellino (1971): The mechanism of the inhibition of plasmin activity by aminocaproic acid. J. Biol. Chem. 246: 4641-4647.

Deutsch, D. G. and E. T. Mertz (1970): Plasminogen: Purification from human plasma by affinity chromatography. Science 170: 1095-1096.

Grant, A. J. (1990): Modifications to the lysine sepharose method of plasminogen purification which ensure plasmin-free Glu-plasminogen. Biochem Int. 20: 519 527.

Hatton, M. W. C. and E. Regoeczi (1974): Some of the observation on the affinity chromatography of rabbit plasminogen. Biochimica et Biophysica Acta 359: 55 56.

Hatton, M. W. C. and E. Regoeczi (1975): The relevance of the structure of lysine bound to sepharose for the affinity of rabbit plasminogen. Biochimica et Biophysica Acta 379: 504-511.

Kline, D. L. (1953): The purification and crystallization of plasminogen (profibrinolysin). J. Biol. Chem. 204: 949-956

Nieuwenhuizen, W. and D. W. Traas (1989):A rapid and simple method for the separation of four molecular forms of human plasminogen. Thromb Haemost 25: 208-210.

Ponting, C. P., J. M. Marshall, and S. A. Cederholm-Williams (1992):Plasminogen: a structural review. Blood Coag Fibrin 3: 605-614.

Silverstein, R. M. (1974): Ion exchange and affinity chromatography during the purification of human plasminogen on sepharose-L-lysine. Thrombosis Research 4: 675-686

Swain,W.R (1998): Plasminogen Assay. Clinical chemistry 14.262-272

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