Transcript Electrophoresisppt

Electrophoresis
Agarose
Gel
Sabrina Schmidtke
Partnership for Environmental Education and Rural Health
Protein Chemistry Laboratory
Texas A&M University
peer.tamu.edu
cerh.tamu.edu
pcl.tamu.edu
What is Electrophoresis?
Electrophoresis is a laboratory
technique for separating mixtures
of charged molecules.
CSI Video Link
• 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.
peer.tamu.edu
cerh.tamu.edu
pcl.tamu.edu
Separation of a Mixture of
Charged Molecules
Charged molecules are separated based on
their electrical charge and size.
Positive Molecules
Analyze
Charge
Separation
Size
Separation
Mixture of
Charged Molecules
Identify
Purify
Negative Molecules
peer.tamu.edu
cerh.tamu.edu
pcl.tamu.edu
Real Life Examples of Uses for
Electrophoresis
• Law Enforcement Agencies
• Hospitals
• Genetics Research
peer.tamu.edu
cerh.tamu.edu
pcl.tamu.edu
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.
peer.tamu.edu
cerh.tamu.edu
pcl.tamu.edu
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.
N
+
O
-
+
+
N
Positively Charged
Amino Acid
peer.tamu.edu
-
+
+
Positively Charged Peptide
cerh.tamu.edu
+ - +
+ - + - + +
- +
Negatively Charged Protein
pcl.tamu.edu
How Separation Occurs
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
peer.tamu.edu
Proteins Entering
Porous Material
cerh.tamu.edu
Smallest Move
Fastest
pcl.tamu.edu
Gel Electrophoresis
Gels can be made from substances such as agarose or
polyacrylamide.
• 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.
Red Sea Weed
• 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
peer.tamu.edu
cerh.tamu.edu
pcl.tamu.edu
Illustration of Gel Electrophoresis
- - Negative Electrode - -
- - Negative Electrode - -
Wells
+ + Positive Electrode + +
+ + Positive Electrode + +
Before Electrophoresis
After Electrophoresis
peer.tamu.edu
cerh.tamu.edu
pcl.tamu.edu
Gel Electrophoresis Experiment
Edible Colors
peer.tamu.edu
cerh.tamu.edu
pcl.tamu.edu
Observing the Gel
peer.tamu.edu
cerh.tamu.edu
All
Blue
Green
Red
Yellow
Possible results.
pcl.tamu.edu
Observing the Gel
peer.tamu.edu
cerh.tamu.edu
Red
Yellow
Green
Blue
Mixed
Red
Yellow
Blue
• 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.
pcl.tamu.edu
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.
peer.tamu.edu
cerh.tamu.edu
pcl.tamu.edu
Alternative Experiments
Skittles
1)
2)
3)
4)
5)
peer.tamu.edu
Grape
Lime
Lemon
Orange
Strawberry
1
cerh.tamu.edu
2
3
4
5
pcl.tamu.edu
Alternative Experiments
Kool-Aid
1)
2)
3)
4)
5)
peer.tamu.edu
Strawberry
Orange
Tropical Punch
Grape
Ice Blue Raspberry Lemonade
cerh.tamu.edu
1
2
3
4
5
6
pcl.tamu.edu