Electrophoresis
description
Transcript of Electrophoresis
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Electrophoresis
Gel
Agarose
Sabrina SchmidtkePartnership for Environmental Education and Rural Health
Protein Chemistry LaboratoryTexas A&M University
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Electrophoresis is a laboratory technique for separating mixtures
of charged molecules.
What is Electrophoresis?
• Mixture: a material composed of two or more elements or parts.
• Charged Molecules: a molecule (such as a protein or DNA) that has too many or too few electrons.
CSI Video Link
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Charged molecules are separated based on their electrical charge and size.
Separation of a Mixture of Charged Molecules
Charge Separation
Size Separation
Analyze
Identify
PurifyMixture of Charged Molecules
Positive Molecules
Negative Molecules
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Real Life Examples of Uses for Electrophoresis
• Law Enforcement Agencies
• Hospitals
• Genetics Research
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Components of Electrophoresis
• Electrical Current – the flow of electric charge• Positive Electrode – the wire that collects electrons• Negative Electrode – the wire that emits electrons• Porous – containing pores, permeable to fluids
and small particles • Sieve – a mesh device to filter small particles out of a
mixture of larger particles.
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How Separation Occurs
Electrical Charge:Many molecules (amino acids, peptides, proteins, DNA, and RNA) have naturally occurring negative and positive charges on them. The sum of these charges determines the overall charge. When introduced to an electrical current, negatively charged molecules are attracted to the positive electrode and positively charged molecules are attracted to the negative electrode.
- +
-+
-
+-
+-
+
- +
- - +
- -
--
Negatively Charged Protein
-+
-+
+
Positively Charged Peptide
+
N
N
O+
Positively Charged
Amino Acid
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Molecule Size:The porous material is made of microscopic particles suspended in a gel. The microscopic particles attach to one another forming tunnels that act as a sieve to separate the molecules. Small molecules can move faster than large molecules.
Porous Material
Proteins Entering Porous Material
Smallest Move Fastest
How Separation Occurs
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• Agarose – a complex sugar chain from red seaweed. It is commonly used in foods (ice cream, whipped cream, and jellies) and many biological mediums. It has a large pore size good for separating large molecules quickly.
• Polyacrylamide – chain of acrylic acid molecules. It is often used to make plastics and rubber. It has a small pore size good for separating small molecules slowly. *Polyacrylamide is a neurotoxin!
Acrylic Acid
Gels can be made from substances such as agarose or polyacrylamide.
Red Sea Weed
Gel Electrophoresis
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Illustration of Gel Electrophoresis
- - Negative Electrode - -
+ + Positive Electrode + +
- - Negative Electrode - -
+ + Positive Electrode + +
Before Electrophoresis After Electrophoresis
Wells
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Gel Electrophoresis Experiment
Edible Colors
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Observing the Gel
Possible results.
Yel
low
Red
Gre
en
Blu
e
All
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• This gel was run for 120 minutes, it shows better separation of the dyes and good replication for the dyes.
• The size of molecules from smallest to largest are: yellow, red, pink, and blue.
Mix
ed
Blu
e
Gre
en
Yel
low
Red
Blu
e
Yel
low
Red
Observing the Gel
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Alternative Experiments
• Other Samples– Separate the food dyes used in Kool-Aid and Skittles.– Separate proteins and DNA. (will require additional materials)
• pH Change– Change the pH of the buffer in the gel and the tank to observe
the changes it makes on the samples.
• Change the Percentage of Agarose Used– Observe how using higher/lower concentrations of agarose will
change the separation of dyes.
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Skittles
1 2 3 4 5
1) Grape2) Lime3) Lemon4) Orange5) Strawberry
Alternative Experiments
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Kool-Aid
1 2 3 4 5 6 1) Strawberry2) Orange3) Tropical Punch4) Grape5) Ice Blue Raspberry Lemonade
Alternative Experiments