Download Biology 164 Laboratory Protein Fingerprinting: Discerning

Biology 164 Laboratory
Protein Fingerprinting: Discerning Evolutionary Relationships Among Animals
Using PAGE (Polyacrylamide Gel Electrophoresis)
Analysis of Muscle Proteins
(Adapted from a BioRad Biotechnology Explorer Kit)
Introduction
In this laboratory experiment we will compare the muscle proteins of a variety of species of marine,
aquatic and terrestrial animals in an attempt to discern the evolutionary relationships that exist among
the species based solely on differences and similarities in their muscle proteins.
Slight variations in the amino acid sequence of a given protein arise whenever mutations occur in the
respective gene that codes for that protein. When a protein has an enzymatic function, its variants are
known as allozymes. The amino acid sequences of allozymes are encoded by homologous genes at
different loci that have diverged over evolutionary time. These protein variants represent molecular
markers that can be useful for understanding evolutionary relationships that exist within populations
and among different species. In fact, protein variants were among the first molecular tools used in
studies by evolutionary biologists. Even though the protein marker approach has been supplanted by
more informative DNA-based approaches, protein markers are among the quickest and cheapest
systems for revealing genetic variation at the molecular level.
Before you begin your experimentation, you and your colleagues at your lab table should consider the
evolutionary relationships among the samples available to you. More information and ideas about these
relationships can be found in the section on fish classification on the last page.
All the samples will be prepared from muscle, so there will be a lot of similarity between the sample
protein profiles simply because they are from the same type of tissue. Nevertheless, there should be
clear differences between samples. You can take advantage of the fact that all the samples are from
muscle tissue, and hence the most abundant proteins in each sample should be predominant muscle
proteins.
Once you have run your protein extracts through the elecrophoresis gel, use the molecular weight
markers (Figs. 1 and 2) to estimate the molecular weights of the predominant “unknown” proteins in
your samples. Do any of these molecular weights correspond to the sizes of known muscle proteins
found in the molecular weight markers? Are these proteins expressed more in one animal compared to
another?
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Fig. 2. Calibrated molecular weights for protein standards
run on Tris-HCl and Bis-Tris gels.
There are many other questions you may consider. Are there proteins present in fish that are absent in
invertebrates and terrestrial animals? Are there differences between aquatic (fresh water) and marine
(salt water) fishes? Between cold water or warm water fishes? How about differences between
sedentary and more active fish? For example, flounder is a bottom-dweller and relatively inactive,
whereas a fish like mackerel is a fast-moving ocean fish.
Muscle tissue is easy to prepare for electrophoresis because, in general, it is low in fat and in tough
connective tissue. This allows the proteins to be extracted easily from the muscle tissue and produces
striking results in a few simple steps.
Once muscle samples have been characterized electrophoretically, the resulting data will be used to
perform a phylogenetic analysis of the samples. We will perform this analysis in lab next week.
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Methods
Protocol for muscle protein extraction and sample preparation
Prepare one tube for each of nine tissues to be analyzed, e.g., catfish, trout, salmon, shark, etc.
1. Label a series of 1.5 ml microcentrifuge tubes, one for each sample being prepared.
2. Add 10 drops of Sample Buffer to each labeled tube and close the lid.
3. Cut a piece of muscle tissue (avoid skin, fat and bones) that weighs 0.15 g or has dimensions of 0.5
x 0.5 x 1.0 cm (about the size of a Tic-Tac™) and transfer into the appropriately labeled
microcentrifuge tube.
4. Flick the microcentrifuge tube 15 times with your finger to agitate the tissue in the buffer.
5. Incubate 5 minutes at room temperature to solubilize proteins.
6. Spin the tubes in the microcentrifuge for about 30 seconds and transfer the supernatant containing
the extracted proteins to another labeled 1.5 ml microcentrifuge tube by pouring the supernatant
from one tube to the other.
Note: It’s not necessary to transfer the entire volume of supernatant. Only a small volume is
actually needed for gel loading.
7. Heat the sample in the second tube for 5 minutes in a 70°C hotblock to denature proteins before
electrophoresis.
8. Samples can be stored at room temperature if gels are loaded within several hours or stored at –
20oC for several weeks.
Protocol for loading and running Nu-PAGE gel:
1. We will be using 4-12% Bis-Tris acrylamide gradient gels and a Novex Xcell II electrophoresis
apparatus. CAUTION: ACRYLAMIDE IS EXTREMELY TOXIC. AVOID TOUCHING IT, AND
WEAR LATEX GLOVES FOR ALL GEL HANDLING PROCEDURES.
2. Use a micropipettor to carefully load the wells with 10 µl samples. Be sure to load the samples in a
way that will allow for the most reasonable analysis. Use Lane One to load 10µl of molecular
weight markers. This will also serve to orient direction of gel during the analysis phase.
3. We will run the gels for about 1 1/2 hours at 170V, until the blue tracking dye reaches the bottom of
the gel.
4. Following the run, turn off the power, unplug the power supply, and carefully disassemble the
electrophoresis apparatus. These gradient gels have no stacking gel, so cut the gel off just below the
wells.
5. Carefully place the gel into a labeled plastic dish containing 150 ml. distilled water and let rinse for
5 minutes. Pour off rinse water and repeat rinse procedure two more times.
6. Add the contents of large test tube containing 20 ml. SimplyBlue SafeStain and 2 ml. 20%NaCl to
dish containing gel. Cover and leave on the rotary shaker overnight.
7. Return next morning and discard staining solution down sink (be careful the gel does not follow
stain down drain!!
8. Add 100 ml of distilled water to gel in dish, cover tightly, and store in fridge.
The various bands on the gel represent differing eletrophoretic mobility of the various proteins
extracted from the muscle samples. We will analyze these data next week in lab.
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Classification of Fishes
The modern fish are divided into the chondrichthyes (cartilaginous) and the osteichthyes (bony) fishes,
with the lampreys and hagfishes in the separate agnatha (jawless vertebrates) class that evolved
separately from the ancestral fishes. The chondrichthyes include the sharks and rays, and the
osteichthyes, a very diverse group, include all other modern fishes. Below you will find brief
descriptions of some of the major fish groups.
Class Agnatha Lampreys and hagfishes are eel-like, jawless fish. They do not have scales or paired
fins. Although lampreys and hagfishes both possess many traits of their ancestral forebears, they are
very different from each other.
Class Chondrichthyes
Sharks, rays, skates, and chimaeras
are members of the class
Chondrichthyes. They have cartilaginous rather than bony skeletons, and do not have swim bladders or
lungs. The skin is thick and does not have true scales. There are about 900 species.
Class Osteichthyes The bony fish, or osteichthyes, are the most diverse class of fish. The class is
characterized by having true scales, paired fins, bony skeletons, and movable rays in their fins and tail.
Osteichthyes are divided into two subclasses, the flesh-finned fish (Sarcopterygians), and the ray-finned
fish (Actinopterygians).
Sarcopterygian (flesh-finned) subclass includes lungfish and coelacanth, the fish most closely
related to modern amphibians, reptiles, birds, and mammals.
Actinopterygian (ray-finned) subclass includes most modern bony fish
Teleostei subdivision is the largest group of bony fishes. Teleosts, are organized in 42 orders
containing over 23,000 species. The evolutionary relationships of some of the important
orders are shown below. The orders (not shown) that include the herrings and anchovies
and the eels are considered specialized offshoots of the teleosts and comprise about 6% of
the subdivision.
Subdivision Teleostei
Superorder Ostariophysi
Order Cypriniformes (minnows, carps)
Order Siluriformes (catfish)
Superorder Protacanthopterygii
Order Esociformes (pikes)
Order Osmeriformes (smelts)
Order Salmoniformes (salmon, trout, whitefish)
Superorder Paracanthopterygii,
Order Gadiformes (cod, hakes, pollock)
Superorder Acanthopterygii
Order Percoidei (perches, snook, basses)
Order Pleuronectiformes (flounders, soles)
Order Perciformes (mackerel, tuna, swordfish)
Sturgeons, bowfins, and gars represent the remainder of the bony fishes, and are considered
evolutionary relics. There are about 50 species of these relic fish extant around the world
today, although there were many more in the past.
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