Laboratory 6-Protein Isolation From Fish and Western Blotting



Laboratory 7-Protein Isolation From Fish and Western Blotting

Proteins are the most important macromolecule in the cell, as they are responsible for almost all cellular functions. They play roles that are as diverse from regulating gene expression, to playing a role in maintaining the proper cellular structure, sensing the environment, as well as acting to mediate communication between neighboring and distant cells. In multi-cellular organisms, as cells differentiate (take on specific functions), the profile of proteins that they express become different. For instance, an epithelial cell will express different proteins, than a neuron, etc. Additionally, certain cell types may contain higher protein content than others. One such cell type that contains high protein content is muscle cells. For locomotion to occur, muscles must contract and relax. The contraction and relaxation is mediated by proteins, specifically actin and myosin. Additionally, several others are important in controlling the contraction and relaxation cycle.

In this laboratory, we will isolate proteins from muscle from a variety of different fish, and we will run two SDS-Polyacrylamide gels. The first polyacrylamide gel will be stained with coomassie blue and the complete protein profiles from the muscle for each fish will be studied. We will then develop an evolutionary cladogram based off of the results of your protein profiles. The second polyacrylamide gel we will run will be further used to perform a Western Blot for myosin.

Learning Objectives:

1. Learn how to isolate protein from animal tissue

2. Learn how to study protein profiles, and develop an evolutionary cladogram

3. Learn how to perform a Western Blot

4. Learn the reasons for either comassie staining a gel, as compared to performing a Western Blot

Experimental Objectives:

1. After protein isolation from a variety of different fishes, develop an evolutionary cladogram based on the protein profiles from your coomassie stained gel

2. Determine the differences in the size of myosin protein from the different fish by performing a Western Blot

Introduction:

In this laboratory section, we will be studying proteins from the muscle of fish. The function of muscle is to allow for all of the important daily movements, from locomotion to breathing. Some muscles are responsible for voluntary movement, such as skeletal muscles, whereas others are responsible for involuntary movements. In order for these movements to occur, the muscles must contract, and a muscle contraction is mediated by proteins. Muscle contractions are mediated by only a small number of proteins. Additionally, these proteins that are involved in contraction, must be expressed at extremely high levels. Therefore, it will be easy for us to perform a characterization of proteins isolated from muscle for a variety of fish species.

Skeletal muscle has a specific structure that is specialized for movement. Each muscle consists of a bundle of many muscle fibers. In turn, each fiber is actually one long multi-nucleated cell that is specialized to perform a contraction. When we look at a muscle fiber at the sub-cellular level, we see that each fiber contains many myofibrils, which span the length of the cell, and are composed of repeating units of sarcomeres. The sarcomere, in turn, is the basic unit of contraction inside a muscle cell, and consists of two different types of filaments: thin filaments and thick filaments. The thin filaments consist of the protein actin and the thick filaments consist of the molecular motor myosin (also a protein). Each myosin molecule contains a head that is bound to ATP. In order for the contraction to occur, the myosin head binds the actin. Then, ATP is hydrolyzed allowing the slide along the actin filament, thus shortening the sarcomere.

Actin and myosin are the major players for a muscle contraction to occur, and are conserved across all animal species. However, other proteins are required for muscle contraction to occur. These proteins are also conserved between most animal species, but the degree of conservation varies, and is generally less than that of actin and myosin. In general, there have been 19 proteins involved in muscle contraction. They are shown in the table below.

Evolutionary and Classification of Fishes (Adapted from Bio-Rad Instructors Manual)

Most fish are contained within the superclass Gnathostoma (jawed vertebrates). Only lampreys and hagfish are outside this group, which do not have a jaw. Interestingly, the hagfish is not a vertebrate (I didn’t know this before writing this lab!) and lampreys are primitive vertebrates. Of the Gnathostoma, they can be divided into two different classes, chondrichthyes (cartilaginous fishes) and Osteichthyes (bony fishes). The Chondrichtyes include sharks and rays, whereas the Osteichtyes include all other modern fishes. Below are the brief descriptions of the major fish groups in order from most ancient to most recently diverged.

Hyperotrti (hagfish) are eel-like jawless fishes, which have a skull and no backbone.

Hyperoartia (lamprey) are eel-like jawless fishes which are primitive vertebrates. They have a single nostril and a sucker-like mouth they use to attach to other fish, as well as rocks.

Chondrichthyes (shark, ray, skate and sawfish) have a cartilaginous skeleton. Their skin is thick and without true scales. They also do not have swim bladders or lungs.

Osteichthyes (coelancanth, tuna and haddock) are bony fishes that have true scales, paired fins and movable rays in their fins and tails. They are divided into two subclasses: Lobe-finned fish (Sarcopterygians) and Ray-finned fish (Actinopterygians).

Sarcopterygians (lungfish and coelacanth)

Actinopterygian is the subclass encompassing most modern ray-finned fish including the chondrostei, semionotiformes and teleosts.

Chondrostei (sturgeon) are considered relic bony fishes. They lack scales on most of the body, have a cartilaginous skeleton and have developed a shark-like heterocercal tiail and a rostrum extending past the mouth.

Semionotiformes (gar) are also ancient fish, they have bony scale and a mainly cartilaginous skeleton

Teleosts (herring, carp and pufferfish) comprise the remainder of bony fishes, and are very diverse. These fish have a bony skeleton.

Acanthomorphia (pollock, bass and sole)

In order to learn more about about the fish, you can go to . This may help you make analyses about the relatedness of the fishes in our laboratory.

Building A Cladogram

A cladogram is used as a way to visualize evolutionary relationships between organisms. A cladogram looks like a series of Y’s, or forks in the road. Each branch on the diagram is called clade, and each fork is called a branch point. Each clade contains organisms that are evolutionarily similar than the rest. When constructing a cladogram, only objective data is used to determine evolutionary relationships. For instance, cladograms can be based off of morphological data, which includes studying structural relationships between organisms, as well as molecular data, such as protein, or even DNA sequences.

Cladograms can be constructed for any group of organisms. Each of the organisms we choose to study when constructing the cladogram will have an evolutionary relationship to the others. Each of the organisms will share some characteristics with some of the others. The more characteristics they share, the closer on the cladogram they will be. Furthermore, those that are most similar may be part of a clade, separate from the others in the study. Based on the protein profile data we collect in this laboratory, we will construct a cladogram based off of the fish species we will study.

Performing a Western Blot

Cells contain many proteins, and when we lyse them, run a polyacrylamide gel and stain it with coomassie blue, we can see the entire protein profile of the cells we are studying. However, when we want to study only a specific protein instead of the whole profile, we run a western blot. In order to run a western blot, one must first choose a tissue, or set of tissues, lyse the cells to make a protein extract. Once the extract is made, then we will run our extract on an SDS-polyacylamide gel. This time, instead of running the gel such that the dye front to the bottom of the gel, and then coomassie staining the gel, we will process the gel in a different manner.

After running the Poly-acrylamide gel, we will move the proteins to a nitrocellulose membrane in order to perform an immuno-detection assay. The proteins, which are negatively charged due to the SDS, will be transferred using an electrical current to the membrane. The proteins will bind the surface of the membrane, creating the Western Blot. Interestingly, the transfer of the proteins from the gel to the membrane is done horizontally. Therefore, the position of the proteins on the Western Blot will be the same as they would be on our SDS-Polyacrylamide gel.

Once the proteins are transferred to the blot, we will then probe the blot for our specific protein of interest. To probe the blot, we will use an antibody that is made to specifically bind specifically to the protein we are interested in studying. In order to start the probing procedure, we will first incubate our blot in powdered milk. Powedered milk also contains many proteins, which coat the surface of the membrane. The coating of the membrane surface will block our antibody from binding non-specifically to other proteins. Once the membrane is blocked, we will then incubate our blot with primary antibody. This is the antibody that will specifically interact with our protein of interest. In this laboratory, we will use an antibody that recognizes the myosin light chain. Once the blot has been incubated with primary antibody for a sufficient amount of time, we will then wash off the primary antibody and then incubate the blot with secondary antibody. The secondary antibody specifically recognizes the primary antibody, and is conjugated to an enzyme. Once the Western Blot has been incubated with secondary antibody for the appropriate amount of time, the secondary antibody is washed off.

In order to visualize our protein on the blot, we will use a colorimetric assay. If our protein is present on the blot, upon doing the colorimetric assay, it will appear as a band (similar to the gel). Since our primary antibody is specific for a single protein, we will most likely see only one band per lane (if the protein is present in that sample). In order to do the colorimetric assay, a colorless substrate is placed on the blot. The enzyme that is conjugated to the secondary antibody has the ability to oxidize the substrate into an insoluble purple precipitate that will leave visible deposits on the membrane at the precise location of the protein of interest. Therefore, if our protein is present in a given lane, we will see a purple band.

Experimental Procedures

A. Extraction of Proteins From Fish Cells

In this section we will obtain samples of tissue from several species of fish, and lyse the cells to make a protein extract.

1. Obtain the appropriate number of 1.5 mL microfuge tubes, and label them appropriately for each fish sample we will study.

2. To each tube, add 250 ul of Laemmli sample buffer to each tube. This is the sample buffer you will use to run your gel. Additionally, this sample buffer contains the detergent SDS, and will be effective in lysing our fish cells for protein extraction.

3. Obtain approximately 0.5g (0.5-1cm of tissue) of muscle tissue from each fish (be sure to avoid the fat) and transfer it to the appropriately labeled microfuge tube containing Laemmli sample buffer.

4. Flick the microtube 15 times with your finger to mix the fish tissue into the sample buffer. Alternatively, the sample can be vortexed for a few seconds.

5. Incubate the samples for 5 minutes at room temperature to extract and solubilize the proteins

6. Pipet the buffer containing the extracted proteins into a new 1.5 mL screw cap tube. Be sure not to transfer the solid fish pieces along with the buffer. These are your fish protein samples (Try to get as much of the buffer as possible, as we will run to gels in this laboratory).

7. Boil your fish protein samples, as well as the purified actin and myosin samples. Additionally, boil your protein standards (ladder) to denature the proteins in preparation for electrophoresis. In this lab we will run two gels, one which will be Coomassie Stained, and the other which will be Western Blotted.

Running the SDS-Polyacrylamide Gels

1. Insert the precast gels appropriately into the gel apparatus. Be sure to remove the plastic tape at the bottom of the gel before mounting it into the apparatus. One gel should go on one side and the other gel should go on the other side.

2. Load the boiled protein samples into the bottom of a well. Therefore, load the gel as it says in the following description. In the second lane, place the Kaleidoscope protein standards, in the third lane place the actin and myosin standard (these will provide a reference for where actin and myosin run on the gel). Use the rest of the lanes to run the fish samples. Be sure to note in your notebook, which fish sample is in each lane.

3. Connect the leads to a power supply (red to red and black to black), and electrophorese the samples until the bromophenol blue dye front has traveled to the very bottom of the gel (200 V for ~ 45 minutes).

C. Gel Number 1: Coomassie Staining

1. After electrophoresis, choose one of the gels. Carefully remove the fragile gel from between the glass plates, and submerge the gel in Coomassie Blue stain. Shake gently on the shaker for at least 30 minutes.

2. Remove the gel from the stain solution and place in Destain I for 15 minutes to 1hr. Remove and put in Destain II 1 - 4 hours until the background is clear. (If you leave it too long in the destain, even the proteins will become destained).

3. Put your destained gel on a piece of saran wrap or in Ziploc bag and photograph it with a digital camera. Include a centimeter ruler in your photograph so that you can easily quantify your measurements. You should put a print of this image into your notebook.

4. Use an image modifying software (eg: Photoshop) to make a grayscale TIF file out of your image and save it as sdsgel.TIF. Save the file somewhere on your computer where you can easily find it when needed.

5. Each band you see on the gel represents a specific protein. Furthermore, each protein runs on the gel according to molecular weight, with larger proteins running shorter, and smaller proteins running longer. Figure out the molecular weights of three proteins by using the following procedure. Based on the protein profiles seen on your gel, note which are similar and which are different. From this information, create a cladogram. Note, the ones that are most similar will fall in the same clade.

D. Gel Number 2: The Western Blot

1. Remove the other gel as described in part C, step 1. Be sure to leave the gel on one of the plates.

2. Using a razor blade, chop the wells off of the top of the gel (this will remove the stacking component of the gel)

3. Equilibrate the gel in blotting buffer for 15 minutes on a rocking platform.

5. Soak all fiber pads from the blotting apparatus in the blotting buffer

6. Obtain a white nitrocellulose membrane and mark it with your initials in a corner that will correspond to the top right corner of the gel when you blot. Soak the membrane in blotting buffer. Also, obtain 2 pieces of Watman paper, and place that in blotting buffer.

7. Make a blotting sandwich by:

a. add 1cm depth of blotting buffer to a container and insert the plastic cassette in the buffer black side down

b. Lay a wet fiber pad on the black side of the cassette

c. Lay one wet blotting paper on the fiber pad and roll out air bubbles

d. Lay gel squarely on blotting paper. Be sure when you do this to avoid any bubbles from forming

e. Lay wet nitrocellulose membrane on the gel. Be sure that where you place your initials corresponds to the upper right hand corner of your gel. Also roll out any bubbles between the membrane and the gel.

f. Lay one wet blotting paper on the membrane and roll out air bubbles

g. Lay a wet fiber pad on top of the blotting paper

h. Close the cassette, and clamp with the white clip

8. Set up the mini-trans blot apparatus with the black side of the cassette next to the black side of the apparatus. Then, fill the apparatus with blotting buffer up to the white clip.

9. Place the lid on the tank, and blot at 20V for 2.5 hrs.

10. Dismantle sandwiches, and place blots in blocker overnight (or until the next week).

D. Probing Our Blots

1. Place blot in blocking solution (powdered milk) for 15 mins.

2. Discard blocking solution and incubate membrane with 10 mL of primary antibody for 15 minutes while on a platform shaker (in the back of the lab).

3. Quickly rinse the membrane in 50 mL of wash buffer, then discard the wash

4. Add another 50 mL of wash buffer for 3 minutes while shaking on a platform shaker

5. Discard the wash and then incubate the membrane with 10 mL of secondary antibody for 10 minutes while shaking on a platform shaker

6. Quickly rinse the membrane in 50 mL of wash buffer and discard the wash

7. Add 50 mL of wash buffer and wash the membrane for 3 minutes while shaking on the platform shaker.

8. Discard the wash

9. Add 10 mL of HRP color detection reagent and incubate 30 minutes either with manual shaking or on a rocking platform. As the reaction proceeds watch for color development.

10. Rince the membrane twice with ddH2O and blot dry with a paper towel. Air dry your blot for 30 mins-1 hr. Place in Saran wrap, and then analyze the blot.

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