Simulated electrophoresis of proteins on polyacrylamide gel with SDS

Detailed instructions

General controls:

Procedure:

	1. Choose an animation speed (so it is comfortably displayed in whatever computer your are using)
	2. Choose an unknown sample
	3. Choose an acrylamide concentration for the gel
	4. Choose 2 or more standards, ticking the checkboxes by their name. You may check their identity and properties pressing the 'Protein Info' button.
	5. Choose a voltage
Add Standard	6. Press the button to load standards into the first well.
Add Sample	7. Press the button to load samples into the second well.
Start Stop	8. Press the buttons to switch current on and start running the electrophoresis, and to switch if off before bands exit the lower end of the gel.

Definition of terms

Acrylamide

The main momomer composing polyacrylamide.

Agarose

A polymer that forms a gel with fairly large pores. It is used as the separation medium in electrophoresis of macromolecules, particularly nucleic acids.

Anode

Positive electrode (to which anions, negatively charged, move).

Bisacrylamide

 The common name for N,N'-methylenebis(acrylamide), a monomer composing polyacrylamide.

Buffer

.

Capillary electrophoresis

.

Catode

Negative electrode (to which cations, positively charged, move).

Convection currents

.

Denaturing agent

A chemical or a physical condition that provoke denaturation of macromolecules.

DTT

Dithiothreitol

threo-2,3-dihydroxy-1,4-butanedithiol. Also known as Cleland's reagent.
See reducing agents.
Reduction reaction of disulphides:

 

Electroosmotic flow

.

Electrophoresis

The separation of molecules in solution by application of an electrical field. Depending on the support used, molecules will separate as a function of a combination of their electrical charge, their size and their shape.

 

Electrophoretic flow

.

Mercaptoethanol

More properly, beta-mercaptoethanol or 2-mercaptoethanol. See reducing agents.

Molecular mass

The mass of a molecule. It is usually stated without units, referred to the atomic mass unit (a.m.u. or dalton, Da), therefore it is more correct saying relative molecular mass (M_r). The term molecular weight is less correct. In the case of macromolecules, particularly proteins, it is common to use molecular mass divided by one thousand, so expressed as kilodaltons (kDa). Finally, the amount of substance the mass of which is, in grams, numerically equivalent to M_r corresponds to one mole of the substance.
Examples:

Molecular mass of anhydrous copper sulphate (CuSO₄) is 159.6 amu = 159.6 Da, or its relative molecular mass is M_r = 159.6. Its molar mass is 159.6 g/mol
Molecular mass of myoglobin is 17183 Da = 17.2 kDa; its relative molecular mass is M_r = 17183. Its molar mass is 17183 g/mol

Native

.

 

Polyacrylamide

A polymer generated from acrylamide and bisacrylamide. It forms a gel with pores smaller than those in agarose gels. It is used as the separating medium in electrophoresis of macromolecules, particularly proteins and nucleic acid fragments.

Primary structure

.

 

Random coil

.

Reducing agents

Chemicals able to reduce disuphide bonds in proteins (-S-S-) to sulphydryl groups (-SH). They allow to completely unfold a protein and to separate the subunits in a multimeric protein; both effects are needed so that the mobility of the protein in SDS-PAGE electrophoresis will be according to its molecular mass. The most common are beta-mercaptoethanol and dihtiothreitol (DTT).

Relative migration

.

Rm

Relative mobility or relative migration.

Quaternary structure

.

Secondary structure

.

Tertiary structure

.

 

SDS

 

Sodium dodecylsulphate

A chemical with properties as detergent, commonly used in electrophoresis (the SDS-PAGE technique) due to its ability to denature proteins , binding them in a constant mass ratio. As a result, protein molecules are fully unfolded and covered up with a uniform negative charge; due to this, their electrophoretic mobility is inversely proportional to the number of their amino acid residues.
Other names: SDS, sodium laurylsulphate.

SDS-PAGE

Sodium Dodecyl Sulphate - PolyAcrylamide Gel Electrophoresis. The detergent SDS is included both in the composition of the gel and of the electrophoresis buffer, typically at a 1% concentration. The main use for this technique is estimation of molecular mass of proteins.

Subunits

.

 

Analysis of results

1. Clicking on each protein band in the gel will display at the top their informaction. Take note of the relative mobility for the band of the unknown protein sample. To estimate the molecular mass of the unknown protein:
2. Press the Plot results button to display the calibration graph.
3. On that plot, position the mouse pointer over the abscissa axis; when the value displayed matches the mobility of the unknown protein (that you wrote down before), press the mouse button. An interpolation line will be plotted and you may read by the ordinates axis the value of the logarithm of molecular mass, and at the top that mass.

Practice exercises

A) Influence of the applied voltage

Load the gel with all standards and any one of the unknown protein samples.

Choose in turn each of the voltages available in the simulator and run the electrophoresis until the tracking dye band (purple colour) approaches the lower end of the gel. Write down in each case the mobility of the standards.

1. Which is the effect of an increase in the voltage applied between both electrodes?

B) Influence of the acrylamide concentration

Load the gel with all standards and any one of the unknown protein samples.

Choose in turn each of the acrylamide concentrations available in the simulator and run the electrophoresis until the tracking dye band (purple colour) approaches the lower end of the gel. Write down in each case the mobility of the standards.

1. Which is the effect of an increase in the acrylamide concentration that forms the gel?

When the acrylamide concentration is increased, some standards separate better from each other, while others are less separated.
What does happen to all of them is they move less (although for the smallest ones the difference is minimal).

2. How can you interpret this result, in terms of the mechanism of separation?

C) Self-assessment of results for molecular mass

Choose one of the unknown protein samples, included in the simulator.

Load that sample in the gel, as well as all the standards.

Run the electrophoresis until the band of the tracking dye (purple colour) approaches the lower end of the gel.

Once finished, measure the mobility of the unknown protein and interpolate that value in the graph, to obtain the relative molecular mass.

To confirm that your results are correct:

D) Determination of quaternary structure

Load the gel in the simulator with all the standards and any one of the unknown protein samples (although you will not use the latter).

Run the electrophoresis until the band of the tracking dye (purple colour) approaches the lower end of the gel.

Once finished, access the graph of relative molecular mass against mobility and solve the following problems:

1. An enzyme is treated with SDS and beta-mercaptoethanol and analysed on a polyacrylamide gel with SDS; two bands are observed with relative mobilities of 0.47 and 0.59.
Thanks to some independent experiments involving size exclusion chromatography and ultracentrifugation, the molecular mass of this enzyme has been determined as 128 000.
Using the calibration line obtained in the simulator, estimate the molecular mass of both subunits of the enzyme and deduce which its quaternary structure is.

2. A protein treated with SDS but without beta-mercaptoethanol is analysed on a polyacrylamide gel with SDS, showing a single band with relative mobility of 0.21.
When the same protein is previously treated with SDS and beta-mercaptoethanol, electrophoresis displays two bands, with mobilities 0.48 and 0.61.
Using the calibration line obtained in the simulator, interpret the results.

3. (Advanced) If the protein is previously treated with trypsin, the same band is obtained in the absence of beta-mercaptoethanol, but four bands in its presence. Explain these results.