Estimation of Three Macromolecules Size by Sephadex Gel filtration (Essay Sample)

New England University
(BCHM 210)
127028956000
Introductory molecular biology and biochemistry 1
Prac report 2
127046990000
Raid Halawani
220094219
Estimation of three macromolecules size by sephadex Gel filtration
Abstract
Gel filtration chromatography is a kind of method used in the separation of macromolecules such as protein, nuclei acids, and polysaccharide. The experiment aim was to investigate how to purify three protein, the proteins were blue dextran, hemoglobin and cytochrome c by the use of G – 100 sephadex column. The separation of these molecules was done according to their molecular weight. There was a variation in estimated and actual molecular sizes of protein molecules under experiment.
Introduction
Gel filtration chromatography is a method applied in purification of biomolecules according to the differences in their specific molecular and chemical properties. These include charge, size, hydrophobicity and ligand specificity (Papa Christodoulou, 2014). Size exclusion Gel filtration chromatography has an important role in the separation of macromolecule, such as protein, polysaccharides, nuclei acids and other biological molecules. This method separates molecules according to their different sizes. The molecules for purification were passed through a sephadex column. The Gel filtration chromatography method was used for its suitability in providing conditions for purification without any disturbance. The medium for purification has columns are packed with porous beads. The mobile buffer filled the pores of the matrix and the gaps of the spherical particles. The matrix was the stationary phase of the chromatogram. Bigger molecules, in this case was blue dextran , moves much faster as compared to the smaller ones since a smaller volume of the solution can enter into the large molecules. Smaller molecules cannot move rapidly since they entered the porous beads and still existed in the aqueous medium (David, 2008). Sephadex has exclusion limits for instance, molecules of sizes like G 100, G150, and G 200, their respective exclusion limit will be 100,000., 150,000., and 200,000. For group separation G-50, G-25 and G-10 sephadex were used. The column for G-100 sephadex can hold a biological molecule that weigh 100 k Daltons. The chromatogram effectivity also depends on the distribution of the pore size. The sample volumes were used to determine the volume of the packed bead. In Gel filtration chromatography changing buffer conditions facilities in refolding the proteins that had been denatured. Hemoglobin and cytochrome c were separated according to their molecular weight, by the use of G-100 sephadex column. They were passed through the column for purification. Blue dextran was the largest protein according to molecular weight which according to measurements was 2,000,000 Da. The experiment hypothesis was that larger protein molecules move rapidly as compared to smaller proteins in an aqueous solution.
Method
The experiment used a mixture containing haemoglobin, cytochrome c and blue dextran. Cytochrome and haemoglobin, had molecular weight less than the barring limit of the column, which emerged at the elution volumes larger than the void volume. The column matrix had prepared by use of 0.1M of phosphate buffer. However, a 10ml measuring cylinder, and 20 numbered centrifuge tubes were prepared and calibrated to help measure 1.0 ml column. The height of the column outlet was adjusted and the reservoir set to flow at a slower rate of at least 0.5 ml per minute. The buffer was placed on top of the column to drain down to the level of the gel until the meniscus disappeared. During the procedure, the buffer level needed to be kept stable and was not allowed to fall below set limit. The liquid which was drained off was discarded. A 0.3 to 0.4 Ml of the mixture was pipetted on the gel but the gel surface was kept undisturbed. When all the above set up was complete, the experiment collected an eluate into a 10 Ml measuring cylinder and the wishing was repeated until the colored band was just moved from the top of the gel beads. The column above the gel beads was carefully filled with 0.1M of phosphate buffer. This was done to ensure that the gel surface was not disturbed during the experiment. The column was repeatedly run with a flow rate of one drop per every four second. Then the first 10ml of the continuous dripping of the eluate was collected in the measuring cylinder and changed into a calibrated centrifuge – tube (tube 1). 1.0 Ml of the eluate in tube one was collected and switched to tube two, and collection of the 1.0 Ml fractions continued until all visible color appeared from the column. The color and intensity of each fraction was observed and recorded using a ‘+’ scale for intensity. By using the phosphate buffer, the 1.0 Ml fractions was diluted to a volume of four Ml .Also 1.0 ml of eluate from the measuring cylinder was diluted into 4 Ml. These diluted fractions was read on the spectronic 20 spectrophotometre, at 650 nm and 540 nm compared with a distilled water bank. The absorption maximum for cytochrome c is 550 nm that for haemoglobin was 540 nm, whilst the absorption maximum for cytochrome c was 550 nm that for haemoglobin was 540 nm, whilst the maximum for blue dextran is 650 nm.
Results
Figure 1, the graph showing different absorbance for the elution volumes.
"ENTER THE IMAGE OF THAT GRAPH HERE FROM YOUR PREVIOUS REPORT JUST COPY PASTE IT HERE.”
...