Transcript Separation Science - PhysicsEd.BuffaloState.edu

Biophysical Chemistry
Gel Electrophoresis
Definition
• Electro = Charge + Phorsesis= Carry
• Electrophoresis = Separation of charged
molecules by differences in their rate of migration in
an electric field.
The Components of The System
• Molecules to be separated
– Proteins
– Nucleic Acids
• Support medium
– Gel (Starch, Polyacrylamide or Agarose)
• Buffer System
– High Buffer Capacity
• DC Power Source
– 50 – 1000 V
Common Support Media for
Biological Molecules
• Proteins
– Native Gel (Acrylamide or Starch)
– Denaturing (SDS) Gel (Acrylamide)
• Nucleic Acids – DNA & RNA
– Agarose Gel
– Acrylamide Gel
Factors that Influence Mobility
• Properties of the Molecules to be separated
– Molecular size (MW)
– Molecular shape
– Molecular charge
• Properties of the System
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Electric field strength (V/cm)
Porosity of the support medium (% S)
Conductivity of the buffer (R)
pH of the buffer
These Factors Interact
• Mobility is proportional to charge/MW.
– Charge is affected by buffer Ph
• Mobility is proportional to field strength.
– Allowable field strength is affected by buffer
conductivity (high conductivity high current heat)
• Mobility is inversely proportional to buffer
conductivity.
Making a Separation
• Electrophoresis systems are designed to
optimize the separation of specific molecule
types based on specfic molecular parameters:
– Nucleic acids: Charge/BP is a constant. Separation
can be based on number of base pairs (given all
molecules have same shape). Larger molecules
move slower due to friction with gel
– Proteins: Charge varies as a function of amino acid
composition and buffer pH. Separation is based on
Charge/MW (shape may also vary). The exact
combination of factors varies for each molecule
Separating Proteins Based on
MW
• The problem associated with protein
separation (too many separation
parameters) has been solved by using
denaturing SDS Gels
– The proteins are heat denatured which makes
them all the same shape (linear)
– The proteins are coated with an ionic detergent
(SDS) which gives all molecules approximately
the same overall negative charge
– Separation is based on MW alone
SDS Polyacrylamide Gel Electrophoresis
(SDS PAGE)
Separation of DNA Molecules in
Agarose Gels
• In most cases the molecules are linear
• The phosphate groups bear negative
charge at neutral pH (2 phosphates/BP)
• Therefore mobility will be based on
number of base pairs/molecule
• The Procedure for making and running
agarose gels is shown in the following
video
Physics Concepts Addressed
• Basic DC circuits and Ohm’s law.
– Ionic strength and conductivity
– Ohmic heating
• The concept of an electrical field.
– Force acting on a charged molecule as a result of an
applied voltage
• Frictional resistance to mobility
– Friction as a function of molecular surface area
• The use of logarithmic paper (or spread sheets).
– Determination of molecular size
Practical Considerations for Teaching Gel
Electrophoresis in the High School
• Availability of materials and equipment
– Science Kit & Carolina both have kits for doing DNA
electrophoresis.
• Cost (Demonstration vs. Hands on)
• Safety issues
– Toxic chemicals – minimal toxicity except for ethidium
bromide stain. Can not be used!!!
– Electrical hazards- 80 V DC is the standard voltage
used in most setups. The power supplies in the kits
are often limited to 100 V. GFI outlets are manditory.
References and Web Sites
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Polyacrylamide Gel Electrophoresis of Proteins and DNA-Modern Bio. Inc
http://www.modernbio.com/polyacrimide.htm
Simple Electrophoresis of dyes
http://gslc.genetics.utah.edu/units/activities/electrophoresis/
Vivoy et al. The physics of DNA Electrophoresis. Contemp Phys. 33 1-40
(1992)
Physics and gel electrophoresis: using terminal velocity to characterize
molecular weight http://www.iop.org/EJ/abstract/0143-0807/19/6/011
Edvoteck http://www.southernscientific.com/apbiology_electrophoresis.asp
Wards http://wardsci.com/search.asp?t=ss&ss=Electrophoresis&x=14&y=8
Nat Cent Biotech Edu.
http://www.ncbe.reading.ac.uk/NCBE/MATERIALS/DNA/baseunit.html
Physics at Work (Biophysics)
http://www.phy.cam.ac.uk/camphy/outreach/physics_at_work_2003/exhibito
r/biophysics.htm
Low toxicity stains http://www.bioscience-explained.org/EN1.2/schollar.html