An experiment was performed to separate a mixture of unknown denatured proteins through the process of PAGE, polyacrylamide gel electrophoresis, (a form of vertical electrophoresis), and estimate the molecular mass of the unknown protein sample through the use of a standard curve plotted using a standard protein ladder; PageRuler ™.
The molecular mass of the proteins is determined through the distance migrated; how far the protein moved through the polyacrylamide gel towards the cathode as a current of 200v ran through it.
From the mixture of unknown proteins, 5 bandings formed and their Mr's were calculated from the standard curve (fig 1);
"Polyacrylamide gel electrophoresis (PAGE) has a major role in protein analysis"
Aim of experiment; to separate a mixture of proteins through the process of SDS PAGE polyacrylamide electrophoresis. Gel electrophoresis is a lab procedure that separates macromolecules (e.g. proteins) through their physical properties and electrical charges (imposed by ionising groups within the macromolecule).
The technique used in this experiment is called SDS PAGE;
- SDS - Sodium dodecyl sulphate
- PAGE - polyacrylamide gel electrophoresis
- Denatured proteins used
This technique ensures that migration distance is a direct function of Mr, by taking other factors that can affect migration distance into account and eliminating them, these are;
Charge; native proteins can vary in electrical charge due to the order and interactions of amino acids resulting in a different nature folding, which can affect rate of migration. Therefore, two proteins with the same Mr but different electrical charges could migrate different distances.
Protein structure; the composition and order of amino acids in proteins lead to them having specific secondary, tertiary and quaternary structures, because of this, two proteins with equal Mr's but with different shapes will have different migration rates.
These two variables (unless controlled) will prevent determining Mr by migration through a medium.
Denaturation ensures every polypeptide has an equal Charge to Mass ratio.
Sample buffer, staining and destaining
In the sample buffer there is;
- SDS - binds to proteins - giving negative charge
- Reducing agent - disrupts disulphide bridges between cysteine residues
- Glycerol - allows samples to be loaded more easily due to being denser than running buffer
- Bromophenol blue - allows monitoring of migration
To visualise the protein banding (staining);
- Coomassie blue - stains proteins
- Acetic acid and methanol - fixes proteins in staining solution
Preparing the electrode
The gasket of the electrode is loaded with a pre-cast gel (ensuring the larger plate faces outwards) and then placed into the clamping frame - which is then placed into the tank. The inner chamber is then filled up to the short plate with running buffer solution (filling the wells with solution also), this ensures the top of the gel is in contact with the cathode. To bring the bottom of the gel into contact with the anode the outer chamber is then filled with running buffer solution.
Loading samples and running gel
10µl of PageRuler ™ protein standard stained with bromophenol blue is pipetted into well 3 of the pre-cast gel and 20µl of unknown denatured sample is pipetted into wells 1 and 5. Both these have been kept on ice, mixed and pulse-span in a microcentrifuge. The lid of the electrode was then placed and was connected to a power source. 200v of constant voltage is selected and the gel is run at this for 20 minutes until bromophenol blue has reached bottom of gel. The gel is then removed from the clamping frame and placed in petri dish and the top corner of well 1 is cut off - for ease of identification of wells later on. The gel is then destained.
The Pageruler™ standard's banding is measured from the bottom of the well to the middle of the band (mm) and a calibration curve is then plotted against each band numbers molecular mass (kDa). Then the migration distances of the unknown protein bands are measured (mm) and recorded.
The PageRuler™'s distance migrated is recorded in a table (fig 2) with each bands migration distance and molecular mass present.
The migration distance of the unknown protein sample bands in each well (1 and 5) is then measured and an average for each band is calculated(fig 3);
With these averages now calculated, the mass for each band of the unknown protein can be calculated using the equation of the 3rd-order polynomial curve present in the PageRuler™'s graph;
y = -0.011x3 + 0.9728x2 - 29.1x + 321.89;
Upon inspection of the standard protein's graph, it is visible that the data points fit the 3rd-order polynomial curve very well - with the coefficient of determination,R2, value of 0.979 (0.98 to 2d.p) showing little variability in the data collected. This means that any values taken by using the curves formula are going be reliable and accurate
Another observation that can be made from the plotted results is that there is not a linear relationship between Mr and distance migrated, this can be explained by looking at the type of polyacrylamide gel used in the experiment;
In this experiment, the polyacrylamide gel used was crosslinked, which gives rise to pores being produced in the gel, causing it to act as a molecular sieve - separating the proteins by size (mass). These pores cause resistance to occur on the proteins, with larger proteins encountering a higher level or resistance than smaller ones - hence why they can be separated by mass.
The crosslinked polyacrylamide gel used also had a gradient down it, with the concentration of polyacrylamide starting at 4% at the top of the gel, and ending in 20% at the bottom. Pore size decreases as concentration of polyacrylamide increases.
This type of gel aids visual analysis greatly as it allows a much more defined banding to occur.
If this experiment was done with a non-gradient gel of 20%, then all the banding would have been very close together near the top of the gel - with little migration occurring. This would be due to there being smaller pores at the top of the gel therefore greater resistance being generated at the top of the gel.
Upon completing this experiment, I think it would be interesting to perform the electrophoresis using a different kind of support medium, such as an inert media, which "provide physical support and minimise convection: separation is based on charge density only"
This experiment showed that proteins can be separated and possibly determined by through vertical SDS PAGE gel electrophoresis by manipulating one of their three variables that affect migration rate; Mass, shape and electrical charge, and eliminating the two others.
- Jones, A., Reed, R. and Weyers, J. Practical skills in biology. Fourth edition. 2007.Pearson Education. Essex.