University of Pittsburgh at Bradford Science In Motion Biology Lab 025
Electrophoresis is a widely used method that separates molecules based upon charge, size and shape. It is particularly useful in separating charged biomolecules such as DNA, RNA and proteins. The samples are normally loaded into a gel matrix of polyacrylamide or agarose. The matrix acts as a molecular sieve, such that smaller molecules move through it more quickly than larger molecules. Since molecules migrate at different rates through the gel, they separate. This allows researchers to determine how many different molecules there are in a sample, how big the molecules are, and what similarities or differences there are between the samples.
In this lab activity, muscle proteins from 5 different species of fish will be extracted, prepared for electrophoresis, run on a polyacrylamide gel, and analyzed. The size of the proteins will be determined and comparisons between the different fish species will be made.
SDS-PAGE: A form of polyacrylamide gel electrophoresis (PAGE) that incorporates the detergent sodium dodecyl sulfate (SDS). Protein samples are pretreated by heating them in the presence of the negatively charged detergent SDS, so that proteins and protein complexes are disrupted, or denatured, and all proteins acquire a uniform charge-to-mass ratio. Proteins then will
migrate at rates dependent only on their molecular weights, without their native 3-dimensional shapes or charges being factors.
Denaturation: The disruption of a protein’s secondary, tertiary, and quaternary structure prior to electrophoresis using heat and SDS. This results in having only the linear chain of amino acids
(the primary structure) to migrate through the matrix. In this lab, the Laemmli sample buffer, which contains SDS, will be used to denature the proteins.
Standards: Molecular weight markers that consist of a mixture of proteins of known molecular weights. These standards can be used to determine the approximate molecular weight of unknown proteins. This lab uses two different sets of standards: 1) prestained Kaleidoscope standards that are visible as they migrate through the gel; and 2) actin and myosin standards as a reference to help identify the major conserved muscle proteins.
Daltons: A measure of molecular weight used for proteins. One Dalton is defined as the mass of a hydrogen atom, which is 1.66 x 10 -24 gram. Most proteins have masses on the order of thousands of
Daltons, so the term kilodalton (kD) is used.
Evolutionary relatedness: All the fish samples will contain heavy bands of myosin (210kD) and actin (43kD). Their presence signifies that all fish have actin and myosin as a primary component of muscle and that both proteins have been conserved throughout evolution. Banding patterns between the different species can be compared to determine relatedness (see attached evolutionary tree).
Adapted from BIO-RAD: Evolutionary Relationship of Fish - Protein Profiling 1

Students will be able to:
1.
extract and denature proteins from fish samples.
2.
load and run proteins in electrophoresis.
3.
stain and analyze the results of their samples.
4.
interpret protein profiles as molecular evidence for evolution.
5.
compare phylogenetic relationships with protein gel data.
*Gloves and aprons should be worn at all times to avoid contaminating the samples with human proteins and to avoid staining of hands and clothing.
**Caution should be used around electrophoresis equipment to avoid electrical shock hazards.
6 - 1.5ml EZ Micro test tube
7 - 1.5ml screw-cap micro test tube water bath at 95 o C
Laemmli sample buffer – 30ml
1 - Disposable 1ml pipet
5 - Fish samples ~1g each
1 - marking pen
1.5ml Laemmli sample buffer scissors to cut fish samples
Actin & myosin standard - rehydrated
Kaleidoscope standards - 500µl
1.
Label one flip-top and one screwcap microtube with the name of each fish sample being prepared for electrophoresis.
2.
Add 250µl of Laemmli Sample buffer to each labeled flip-top tube.
3.
Obtain a piece of each fish muscle sample. Cut each to ~ 0.25 x 0.25 x 0.25 cm 3 .
Avoid skin, fat, and bones. Transfer each fish sample to the appropriately labeled flip-top microtube and close the lid.
4.
Gently flick the microtubes 15 times with finger to agitate the tissue in the sample buffer.
5.
Incubate samples for 5 minutes at room temperature to extract and solubilize the proteins.
6.
Carefully pour the sample buffer containing the extracted proteins, but not the solid fish piece, into the correctly labeled screwcap microtube.
7.
Heat the fish samples and the actin and myosin standard (AM) for 5 minutes at
95 o C to denature the proteins.
8.
Store samples in freezer until next class.
Adapted from BIO-RAD: Evolutionary Relationship of Fish - Protein Profiling 2

Fish protein extracts (Day 1)
7- pipet tips for gel loading
Mini PROTEAN 3 module
1-20µl automatic pipet precast gel, 15% - 10 well sample loading goad – 10 well
Power supplies
1x Tris-glycine-SDS ~350ml/station
Bio-Safe Coomassie protein stain staining trays water bath distilled water for destaining thin metal weighing spatula actin & myosin standard
Kaleidoscope standard
Note: electrophoresis gels and gel boxes have been prepared for you
Step 1: Load and run electrophoresis gels
1.
Place a yellow sample loading guide on the top of the electrode assembly. The guide will direct the pipet tip to the correct position for loading each sample in the well.
2.
Load the samples into the wells following this guide:
Lane
1
2
3
4
5
6
7
8
9
Volume empty empty
10 µl
10 µl
10 µl
10 µl
10 µl
10 µl
10 µl
Sample none none
Kaleidoscope
Fish 1
Fish 2
Fish 3
Fish 4
Fish 5
AM standard
10 empty none
3.
To load each sample, use a thin gel loading micropipet tip to withdraw 10 µl of each protein sample from its tube and gently transfer it into the designated well.
4.
After loading all samples, remove the sample loading guide, place the lid on the tank, and insert the leads into the power supply, matching red to red and black to black.
5.
Run gel for 30 minutes at a constant voltage of 200V.
Adapted from BIO-RAD: Evolutionary Relationship of Fish - Protein Profiling 3

Step 2: Stain gels
**Wear gloves when disassembling and staining the gels to avoid contaminating the gels with human proteins and to avoid staining your skin.**
1.
When the gels are finished running, discard the buffer from the inner chamber, release the cams, and remove the gel cassettes from the assembly.
2.
Lay each gel cassette flat on the bench with the short plate facing up. Cut the tape along the sides of each gel cassette. Carefully pry apart the gel plates using your fingertips or a weighing spatula. The gel will adhere to one of the plates.
3.
Transfer the plate with the gel to a staining tray containing Bio-Safe Coomassie stain. The stain will cause the gel to detach from the plate.
4.
Stain the gels for 1 hour. Gentle agitation throughout staining time gives best results.
Step 3: Destain gels
1.
After staining for 1 hour, pour off stain and return to bottle.
2.
Destain the gels in a large volume of water, changing it several times if possible.
Allow gels to destain overnight for best results.
**After destaining, blue bands should be distinct on a clear background. Gels can be stored in water for up to one week before drying.
DAY 1
1.
What is SDS-PAGE?
2.
Explain the importance of treating the samples with heat and SDS prior to electrophoresis.
Adapted from BIO-RAD: Evolutionary Relationship of Fish - Protein Profiling 4

DAY2
1.
What happened to the proteins in your loaded samples when you turned on the electric current?
2.
Why were you able to see the different colored Kaleidoscope standard proteins while the gel ran?
3.
What must happen to make the proteins in your samples become visible?
Most similar species
1.
In your pre-lab activity, which two fish species did you list as being most related?
__________________________ and _______________________
2.
Of all the muscle proteins that you found in these two species, how many are present in both species? _______
3.
What is the total number of different kinds of proteins that you were able to detect on your gel, in these two species? _______
4.
Of the total number of proteins in this pool, how many are found in common to both species listed in question 1 above?
(number of proteins in question 2)
(number of proteins in question 3) X 100 = _______%
Adapted from BIO-RAD: Evolutionary Relationship of Fish - Protein Profiling 5

Least similar species
5.
In your pre-lab activity, which two fish species did you list as being least related?
__________________________ and _______________________
6.
Of all the muscle proteins that you found in these two species, how many are present in both species? _______
7.
What is the total number of different kinds of proteins that you were able to detect on your gel, in these two species? _______
8.
What percent of muscle proteins were common to these least similar species?
_______%
9.
Do your data support the relationships indicated by the phylogenetic tree?
Interpretation of individual bands
1.
What do the relative positions of the bands on the gel indicate about the proteins in the bands?
2.
Are all the bands of equal thickness?
3.
How would you explain the observation that some proteins form thin bands while others form thick bands?
Adapted from BIO-RAD Biotechnology Explorer TM Protein Fingerprinting Instruction manual
Adapted from BIO-RAD: Evolutionary Relationship of Fish - Protein Profiling 6