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.

http://www.dnalc.org/ddnalc/resources/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

(directio

n o

f co

nven

tion

al curren

t)

Electro

n flo

w

typical gels

+ +

- -

Movement of biomoleculesDNA, RNA, Proteins

• Strong electric field → large force moves molecules faster – For nucleic acids (DNA and RNA), average

voltages range from ~80-200 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:

DNAExternal Phosphates give DNA a netNegative 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 ratioDecreases.

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+FElectric Ffriction

+ anode

- cathode

Force diagram

Biomolecules migrate at Terminal velocity

∑F = FE + Ff = ma = 0

Who can resist a little Physics?:

2H+ + 2e-→H2(gas)

e-

+ anode

- cathode

Electrolysis Water isH20 → H+ + OH-

2H2O→ O2(gas) + 4H+ + 4e-

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.

A little Chemistry:

Electrophoresis LAB1.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 #1 792.85 g/mol Crystal Violet

408.0 g/mol

Fluorescein 376.27 g/mol

Red #40 496.42 g/ mol

Yellow #5 452.38 g/mol

Safranin 350.85 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)