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A tool for s eparating 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.

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slide1

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

Electrophoresis

slide2
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
The Gel Substance
  • Agarose (from seaweed)
  • Polyacrylamide
    • Powders that gel when boiled in liquid and cooled to room temp.
the buffer
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
THE ELECTRIC FIELD

Parallel Electrodes

- cathode

Electric field (direction of conventional current)

Electron flow

+ anode

movement of biomolecules dna rna proteins
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
slide8

Goal:

  • Maximize the separation of molecules of different molecular weights
  • Minimize changes in electrolyte conductivity
  • Don’t overheat (melt) the gel
slide9

Physical features of biomolecules affect their ability to be separated from each other:

  • Size (molecular weight)
  • net charge
  • Conformation (shape)
slide10

Size and Net Charge

External Phosphates give DNA a net

Negative charge.

DNA

-

Longer molecule means

a lot more atoms and

a few more negative charges.

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-

-

-

-

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As length increases, charge/mass ratio

Decreases.

-

-

-

-

-

-

Larger molecules go slower

slide11

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.

slide12

FElectric

Ffriction

Q+

∑F = FE + Ff = ma = 0

Biomolecules migrate at Terminal velocity

Who can resist a little Physics?:

Force diagram

+ anode

- cathode

slide13

Electrolysis

Water is

H20 → H+ + OH-

+ anode

- cathode

e-

e-

2H+ + 2e-→H2(gas)

2H2O→ O2(gas) + 4H+ + 4e-

A little Chemistry:

  • 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.
electrophoresis lab
Electrophoresis LAB
  • Characterize physical features of dye molecules (relative size, charge, relative amount)
  • Determine whether dyes are pure (only 1 color) or complex (more than one color)
  • Identify the 2 dyes in an unknown mixture
these are some of the individual colors in your dyes
These are some of the individual colors in your dyes

Crystal Violet

408.0 g/mol

Red #40

496.42 g/ mol

Blue #1

792.85 g/mol

Fluorescein

376.27 g/mol

Yellow #5

452.38 g/mol

Safranin

350.85 g.mol

terminology
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)