An experiment was performed to divide a mixture of unknown denaturized proteins through the procedure of PAGE, polyacrylamide gel cataphoresis, ( a signifier of perpendicular cataphoresis ) , and gauge the molecular mass of the unknown protein sample through the usage of a standard curve plotted utilizing 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 strias formed and their Mr ‘s were calculated from the standard curve ( fig 1 ) ;
“ Polyacrylamide gel cataphoresis ( PAGE ) has a major function in protein analysis ”
Aim of experiment ; to divide a mixture of proteins through the procedure of SDS PAGE polyacrylamide cataphoresis. Gel cataphoresis is a lab process that separates supermolecules ( e.g. proteins ) through their physical belongingss and electrical charges ( imposed by ionizing groups within the supermolecule ) .
The technique used in this experiment is called SDS PAGE ;
- SDS – Sodium dodecyl sulfate
- PAGE – polyacrylamide gel cataphoresis
- Denatured proteins used
This technique ensures that migration distance is a direct map of Mr, by taking other factors that can impact migration distance into history and extinguishing them, these are ;
Charge ; native proteins can change in electrical charge due to the order and interactions of amino acids ensuing in a different nature folding, which can impact rate of migration. Therefore, two proteins with the same Mr but different electrical charges could migrate different distances.
Protein construction ; the composing and order of amino acids in proteins lead to them holding specific secondary, third and quaternate constructions, because of this, two proteins with equal Mr ‘s but with different forms will hold different migration rates.
These two variables ( unless controlled ) will forestall finding 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 Bridgess between cysteine residues
- Glycerol – allows samples to be loaded more easy due to being denser than running buffer
- Bromophenol blue – allows monitoring of migration
To visualize the protein stria ( staining ) ;
- Coomassie blue – discolorations proteins
- Acetic acid and methanol – fixes proteins in staining solution
Fixing the electrode
The gasket of the electrode is loaded with a pre-cast gel ( guaranting the larger home base faces outwards ) and so placed into the clamping frame – which is so placed into the armored combat vehicle. The interior chamber is so filled up to the short home base with running buffer solution ( make fulling the Wellss with solution besides ) , this ensures the top of the gel is in contact with the cathode. To convey the underside of the gel into contact with the anode the outer chamber is so filled with running buffer solution.
Loading samples and running gel
10µl of PageRuler ™ protein standard stained with bromophenol blue is pipetted into good 3 of the pre-cast gel and 20µl of unknown denaturized sample is pipetted into Wellss 1 and 5. Both these have been kept on ice, assorted and pulse-span in a microcentrifuge. The palpebra of the electrode was so placed and was connected to a power beginning. 200v of changeless electromotive force is selected and the gel is run at this for 20 proceedingss until bromophenol blue has reached underside of gel. The gel is so removed from the clamping frame and placed in petri dish and the top corner of good 1 is cut off – for easiness of designation of Wellss subsequently on. The gel is so destained.
The Pageruler™ criterion ‘s stria is measured from the underside of the well to the center of the set ( millimeter ) and a standardization curve is so plotted against each set Numberss molecular mass ( kDa ) . Then the migration distances of the unknown protein sets are measured ( millimeter ) and recorded.
The PageRuler™ ‘s distance migrated is recorded in a tabular array ( fig 2 ) with each sets migration distance and molecular mass nowadays.
The migration distance of the unknown protein sample sets in each well ( 1 and 5 ) is so measured and an norm for each set is calculated ( fig 3 ) ;
With these norms now calculated, the mass for each set of the unknown protein can be calculated utilizing the equation of the 3rd-order multinomial curve nowadays in the PageRuler™ ‘s graph ;
Y = -0.011×3 + 0.9728×2 – 29.1x + 321.89 ;
Upon review of the standard protein ‘s graph, it is seeable that the information points fit the 3rd-order multinomial curve really good – with the coefficient of finding, R2, value of 0.979 ( 0.98 to 2d.p ) demoing small variableness in the information collected. This means that any values taken by utilizing the curves expression are traveling be dependable and accurate
Another observation that can be made from the aforethought consequences is that there is non a additive 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, doing it to move as a molecular screen – dividing the proteins by size ( mass ) . These pores cause opposition to happen on the proteins, with larger proteins meeting a higher degree or opposition than smaller 1s – hence why they can be separated by mass.
The crosslinked polyacrylamide gel used besides had a gradient down it, with the concentration of polyacrylamide get downing at 4 % at the top of the gel, and stoping in 20 % at the underside. Pore size lessenings as concentration of polyacrylamide additions.
This type of gel AIDSs ocular analysis greatly as it allows a much more defined stria to happen.
If this experiment was done with a non-gradient gel of 20 % , so all the stria would hold been really near together near the top of the gel – with small migration happening. This would be due to there being smaller pores at the top of the gel hence greater opposition being generated at the top of the gel.
Upon finishing this experiment, I think it would be interesting to execute the cataphoresis utilizing a different sort of support medium, such as an inert media, which “ supply physical support and minimise convection: separation is based on charge denseness merely ”
This experiment showed that proteins can be separated and perchance determined by through perpendicular SDS PAGE gel cataphoresis by pull stringsing one of their three variables that affect migration rate ; Mass, form and electrical charge, and extinguishing the two others.
- Jones, A. , Reed, R. and Weyers, J. Practical accomplishments in biological science. Fourth edition. 2007.Pearson Education. Essex.