A tool for separating specific biomolecules from a mixture (A key step in most biotechnology...
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Transcript of A tool for separating specific biomolecules from a mixture (A key step in most biotechnology...
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
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.
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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