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Gel Electrophoresis
• Technique used for separation of
– Deoxyribonucleic acid (DNA)
– Ribonucleic acid (RNA)
– Protein molecules
• Using an electric current applied to a gel
matrix
Gel Electrophoresis
• Uses electricity to separate charged molec. on a
gel slab
– Separation based on size, shape and charge
• Gel:
– Powdered agarose (carb. derived from seaweed)
– Dissolve in boiling buffer soln.
– Most common agarose is polyacrylamide (PAGE)
– Gel solidify and placed in get box and covered with
buffer soln.
Gel Electrophoresis
Gel Electrophoresis
Separation
• “Gel” refers to matrix used to contain, then separate
target molecules
– Gel is a crosslinked polymer whose composition and porosity is
chosen based on specific weight and composition of target to be
analyzed
• "Electrophoresis" refers to electromotive force (EMF)
that is used to move molecules through gel matrix
– By placing molecules in wells in gel and applying an electric
current, molecules will move through matrix at different rates
– Usually determined by mass
• Toward the positive anode if negatively charged
• Toward the negative cathode if positively charged
Visualization
After Electrophoresis Is Complete….
Smears (thousands
of different size
molec in small
concentration)
No nucleic acids
DNA so large
will not load
Ex: eukaryotic
genome
Proteins
• Companies that produce protein products
or study proteins must be able to:
– Separate the protein of interest
– Determine that amount of protein present.
• Characteristics of proteins that make it
possible to achieve either one of both
points above:
– Overall charge, size, shape, and solubility
Proteins
• SDS-PAGE
– Gel electrophoresis allows for the separation
of proteins based on charge, size, and shape.
– Polyacrylamide gel electrophoresis is utilized
(PAGE).
• Allows for better resolution
• 4-18% gels most commonly used
– Higher concentration for smaller proteins
• When protein size unknown gradient gels can be
used.
– Less concentrated at the top than the bottom
Proteins
• SDS-PAGE
– Use of sodium dodecyl
sulfate (SDS)
• Denatures proteins into
polypeptide strands
• Gives each polypeptide
strand an overall negative
charge
• Proteins studied are
strictly being separated by
size
Proteins
• SDS-PAGE
– Visualization of proteins in
gel
• Coomassie Blue
– Milligram amounts of
protein.
• Silver stain
– Microgram amounts of
protein.
– Size of unknown bands
can be determined from
comparison to protein
molecular weight standard
This project is funded by a grant awarded under the President’s Community Based Job Training Grant as implemented
by the U.S. Department of Labor’s Employment and Training Administration (CB-15-162-06-60). NCC is an equal
opportunity employer and does not discriminate on the following basis:
•against any individual in the United States, on the basis of race, color, religion, sex, national origin, age disability,
political affiliation or belief; and
•against any beneficiary of programs financially assisted under Title I of the Workforce Investment Act of 1998 (WIA), on
the basis of the beneficiary’s citizenship/status as a lawfully admitted immigrant authorized to work in the United
States, or his or her participation in any WIA Title I-financially assisted program or activity.