A tool for separating specific biomolecules from a mixture (A key step in most biotechnology applications) Electrophoresis

Electrophoresis: movement of charged particles through a substance when an electric field is applied. rces/electrophoresis.html

The Gel Substance Agarose (from seaweed) Polyacrylamide –Powders that gel when boiled in liquid and cooled to room temp.

The Buffer Ions in solution –Conduct the current from the electrodes through the gel –Maintain pH so that biomolecules keep their original charge

THE ELECTRIC FIELD + anode - cathode Parallel Electrodes Electric field (direction of conventional current) Electron flow

typical gels

Movement of biomolecules DNA, RNA, Proteins Strong electric field → large force moves molecules faster –For nucleic acids (DNA and RNA), average voltages range from ~ Volts DC The more concentrated the gel matrix, the slower the migration

Goal: Maximize the separation of molecules of different molecular weights Minimize changes in electrolyte conductivity Don’t overheat (melt) the gel

Size (molecular weight) net charge Conformation (shape) Physical features of biomolecules affect their ability to be separated from each other:

DNA External Phosphates give DNA a net Negative charge Longer molecule means a lot more atoms and a few more negative charges. Larger molecules go slower Size and Net Charge As length increases, charge/mass ratio Decreases.

Conformation (shape) DNA can be supercoiled - goes faster than when it is linear Proteins can take on many different 3-D shapes – These are denatured in the gel so that they are all essentially linear.

Q+ F E lectric F frictio n + anode - cathode Force diagram Biomolecules migrate at Terminal velocity ∑F = F E + F f = ma = 0 Who can resist a little Physics?:

2H + + 2e - → H 2(gas ) e-e- + anode - cathode Electrolysis Water is H 2 0 → H + + OH - 2H 2 O→ O 2(gas) + 4H + + 4e - e-e- The generated amount of hydrogen is twice the amount of oxygen. Both are proportional to the total electrical charge that was sent through the water.proportionalelectrical charge A little Chemistry:

Electrophoresis LAB 1.Characterize physical features of dye molecules (relative size, charge, relative amount) 2.Determine whether dyes are pure (only 1 color) or complex (more than one color) 3. Identify the 2 dyes in an unknown mixture

These are some of the individual colors in your dyes Blue # g/mol Crystal Violet g/mol Fluorescein g/mol Red # g/ mol Yellow # g/mol Safranin g.mol

Terminology Pure dye has 1 color Complex dye has more than one color in it. Your unknown is made of 2 dyes (may be complex, pure or one of each)