Académique Documents
Professionnel Documents
Culture Documents
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.
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
Analyze
Size Separation
Charge Separation
Identify
Purify
pcl.tamu.edu
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
N
Positively Charged Amino Acid
peer.tamu.edu
Porous Material
peer.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.
peer.tamu.edu
cerh.tamu.edu
pcl.tamu.edu
Wells
+ + Positive Electrode + +
+ + Positive Electrode + +
Before Electrophoresis
peer.tamu.edu cerh.tamu.edu
After Electrophoresis
pcl.tamu.edu
peer.tamu.edu
cerh.tamu.edu
pcl.tamu.edu
peer.tamu.edu
cerh.tamu.edu
pcl.tamu.edu
Materials List
Chamber and Power Supply
Plastic dish Slide box Aquarium sealant Large needle Seizing wire Hot glue gun and glue sticks Nine-volt batteries Nine-volt battery clip Alligator clips Scissors
peer.tamu.edu
cerh.tamu.edu
pcl.tamu.edu
Materials List
Samples and Gel Preparation Small test tubes/vials
peer.tamu.edu
Tube rack Permanent marker Transfer pipettes Capillary tubes with bulb Food colors 50% glycerol solution 2 x 3 glass slide Well comb Tris-Borate-EDTA Buffer (TBE) Agarose Graduated cylinder Erlenmeyer flask Balance Microwave or hot water bath
cerh.tamu.edu pcl.tamu.edu
Safety Precautions
Chemicals Aquarium Sealant, Agarose, and
TBE Buffer are all irritants. If you get them on your skin or in your eyes, rinse with water. Electricity do not touch the alligator clips or the buffer when the power supply is assembled and hooked up. IT WILL ELECTROCUTE YOU!!!! Hot Objects the hot glue gun and the agarose solution will both be hot. If you get burned, rinse the burn with cool water and seek medical attention if necessary. Glassware (beakers, graduated cylinders, slides) If anything is broken dispose of the glassware in an appropriate manner. Sharps the needle, seizing wire ends, and capillaries are all sharp. Be careful when handling these items and dispose of them properly.
peer.tamu.edu
cerh.tamu.edu
pcl.tamu.edu
peer.tamu.edu
cerh.tamu.edu
pcl.tamu.edu
peer.tamu.edu
cerh.tamu.edu
pcl.tamu.edu
peer.tamu.edu
cerh.tamu.edu
pcl.tamu.edu
peer.tamu.edu
cerh.tamu.edu
pcl.tamu.edu
peer.tamu.edu
cerh.tamu.edu
pcl.tamu.edu
peer.tamu.edu
cerh.tamu.edu
pcl.tamu.edu
peer.tamu.edu
cerh.tamu.edu
pcl.tamu.edu
peer.tamu.edu
cerh.tamu.edu
pcl.tamu.edu
peer.tamu.edu
cerh.tamu.edu
pcl.tamu.edu
Red
Green
Blue
peer.tamu.edu
cerh.tamu.edu
Yellow
pcl.tamu.edu
All
peer.tamu.edu
cerh.tamu.edu
Yellow
pcl.tamu.edu
Mixed
Red
Green
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.
Mixed Green Blue Red Blue
peer.tamu.edu
cerh.tamu.edu
Yellow
Yellow
pcl.tamu.edu
Red
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.
peer.tamu.edu
cerh.tamu.edu
pcl.tamu.edu
Alternative Experiments
Skittles
1) 2) 3) 4) 5)
peer.tamu.edu
cerh.tamu.edu
pcl.tamu.edu
Alternative Experiments
Kool-Aid
1) 2) 3) 4) 5)
peer.tamu.edu
cerh.tamu.edu
pcl.tamu.edu