LECTURE 7 - ELECTROPHORESIS

What is the general concept of electrophoresis

Electrophoresis is a separation technique that occurs based on the migration of ions in solution under the influence of an electric field.

What causes the steady ion speed in electrophoresis

When the accelerating force and the frictional force equal each other

What is the electrophoretic mobility

it is the constant of proportionality between the speed of the ion and the strength of the electric field.

What is the relationship between electrophoretic mobility and charge

It is proportional: think when charge is higher that means it goes faster so the constant is higher

What is the relationship between electrophoretic mobility and friction coefficient

They are inversely proportional think when a molecule is larger… it obviously has a larger surface and therefore friction which will result in a lower electrophoretic mobility constant.

How do you improve the resolution in electrophoresis techniques

It is improved by decreasing the width of the chamber, this reduces the stray away from the ion and the variability of mouvement because there is less place, this results in a narrower peak because the retention times end up being closer together.

Elaborate on polyacrylamide gel electrophoresis

It stands for PAGE, in page analytes are seperated by their electrophoretic mobility either by charge number or overall size.

What is polyacrylamide

It is a gelatinous-like material used to seperate proteins and DNA

Elaborate on PAGE of DNA

The DNA backbone is negatively charged therefore the DNA when placed on the PAGE is seperated by the charge/size ratio where the friction dominates and larger fragments move through more slowly. Overall tho DNA cannot be seen so it must be stained with ethidium bromide

What do you stain DNA with in PAGE

To be able to see DNA you need to add ethidium bromide to be able to visualize it

Elaborate on SDS-PAGE for protein

In SDS-PAGE we are seperating proteins. This however is more difficult than DNA since proteins varry in charge size and composition. Therefore to be able to seperate it accordingly SDS need to be applied to the protein as it will coat the protein in a negatively charge as well as denature it as rendering a completely linear complex so it can be accurately seperated by size. It is important to add a protein ladder as a reference for sample size and also add bromophenol blue to sample as it acts as a tracking dye to see how far it goes. Sometime proteins cannot be visualized without stain (if not aromatic) so you need to have coomasie blue dye. Small proteins are less resistant and go faster

What is SDS

an anionic surfactant that coats the protein with negative charges so that proteins can be seperated by size

What is the use of Coomasie blue or silver

To visualize band in SDS-PAGE

What is the role of Bromophenol blue

It is the tracking dye as it migrates ahead of the proteins in the gel

What is a 2D page

a 2D page is able to seperate analytes based on charge/size ratio as well as pH.

What is the isoelectric point

pH when its a neutral charge

zwitterion

Form when the net charge is 0, negative / positive cancel out