In a previous class period you treated bacterial cells with an inducer, IPTG, to turn on the expression of a foreign gene, carried on a plasmid, in these cells. Next, you will analyze the proteins in extracts of the induced cells to determine whether the protein encoded by the foreign gene was made by the bacterial cells. You will prepare extracts by treating the cells with a buffer that will break them open and release their contents. The extract will contain, besides proteins, everything else that is in the cell. You will centrifuge the extracts to remove insoluble materials, then load the cleared solution onto SDS-polyacrylamide gels. Proteins are generally separated on polyacrylamide, rather than on agarose gels, because proteins are smaller than DNA molecules. Polyacrylamide gels have smaller "pores" than do agarose gels, and so they do a better job of resolving protein molecules. The details of how SDS polyacrylamide gels differ from other kinds of gels that you are familiar with will be discussed in class.
Like DNA molecules, most protein molecules are colorless, so their positions on a gel must be determined by staining the gel in some way. In this lab, you will stain the gels using a simple and rapid Coomassie blue staining protocol (you will receive a handout with the composition of the staining and destaining solutions, and you will prepare these solutions while the gel is running). Just as with agarose gels for DNA fragment analysis, a set of standards (protein markers of known sizes) is run alongside the samples to help you estimate the sizes of the proteins that you see.
Focus question
What do you expect to see in each lane of the stained protein
gel?
A. SDS-Polyacrylamide Gel Electrophoresis
1. Take bacterial cell pellets out of the freezer and thaw them on ice.
2. To each thawed sample add 100 µL of SDS-sample buffer and resuspend pellet thoroughly. Cap tubes securely.
Leave samples on ice while you set up the gel apparatus. For this experiment you will be provided with gels that have already been poured. You will need to set them up in the gel apparatus and add buffer to make them ready to load. You will be shown how to put the gel apparatus together in class.
3. Set up the gel apparatus, add buffer and check to ensure that there are no leaks. Place the gel box near a working power supply.
4. Using a syringe needle poke a hole in the cap of each tube from step 2, then place the tubes in a water bath or heating block at 95º C for 5 minutes.
The reason for poking a hole in the caps is to allow heated air to escape from the tube. If this is not done, the caps may pop open spraying innocent bystanders with the contents of the tube.
5. Remove tubes from water bath and centrifuge for 5 minutes at 5000 rpm.
Lysis of the cells can release large amounts of bacterial chromosomal DNA which makes the solution viscous and difficult to pipet. Centrifuging helps to pellet the cell wall debris and other high molecular weight components in the extract.
6. Load 10 µL of each sample onto the gel, being careful to avoid the pellet at the bottom of the tube.
7. Load the protein marker supplied to you alongside the sample lanes.
8. Connect the gel box to the power supply and turn the current to 20 milliamps.
Note that in contrast to DNA gels, protein gels are generally run at constant current.
9. Run the gel till the blue dye reaches the bottom. This should take 45 minutes to an hour.
10. Turn off the power, disconnect the cables and remove the gel sandwich from the buffer tank.
B. Staining the gel
1. Pry apart the gel plates with the help of a razor blade.
2. Place the gel in a Tupperware container and add to the container 50 mL of staining solution.
3. Loosely cover the container with its lid, then place it in the microwave oven and heat it on full power for 1.5 to 2 minutes.
Do not cover the container with a tight lid. You will be boiling solutions in the container and must allow for the escape of steam.
4. Remove the container from the microwave, and place it on a rocker platform and shake for 5 minutes to cool the solution.
Be very careful when removing the solution from the microwave. The solution will be very hot and the microwave will be full of vapors of isopropanol and acetic acid which are not pleasant to inhale. This precaution applies to all the steps where you take the heated solutions out of the microwave.
5. Discard the staining solution into a marked waste container, then quickly rinse the gel with distilled water.
6. Pour off the water, then add 50 mL of destaining solution 1.
7. Place container, loosely covered, in the microwave, heat for 1 minute approximately.
8. Discard the solution into a marked waste container, then quickly rinse the gel with distilled water.
9. Pour off the water, then add 50 mL of destaining solution 2.
10. Place container, loosely covered, in the microwave, heat for 1 minute approximately.
11. Place a Kimwipe into the solution to absorb excess dye and let the solution cool to room temperature by shaking it gently on the rocker platform.
12. Observe the gel to determine whether further destaining is desired. If not, the gel may be dried or photographed at this point. If further destaining is required, the gel may be left in the destaining solution for another 15 minutes, with gentle rocking.
In the last class period, you learned how to use SDS-polyacrylamide gel electrophoresis to analyze the proteins in extracts of bacterial cells. The method that you used to visualize the proteins in the gel was a rapid Coomassie Blue staining protocol. Because Coomassie Blue is a non-specific dye that binds to any protein that it encounters it is quick and useful when you want to be able to see all the protein bands in a given mixture. By using the molecular weight markers run alongside, you can also identify the sizes of the proteins you see.
However, you cannot use Coomassie Blue, or any other non-specific stain, to tell you if the protein of your interest is present in an extract. That is, if the mass of your protein of interest is 40 kD and you see a band on a Coomassie stained gel that looks to be 40 kD, you cannot be sure that it is the protein you want, because there may be other 40 kD proteins in the extract that will stain just as well with the dye.
The method of choice when you want to identify a particular band on a gel as representing a particular protein is the western blot. In this method, the proteins in the mix are separated on a gel, just as before, but they are then transferred from the gel to a nitrocellulose membrane. This transfer is achieved by placing the nitrocellulose membrane in contact with the surface of the gel after the run is completed. The proteins may then be allowed to either diffuse out of the gel onto the membrane, or they may be induced to leave the gel and migrate onto the membrane by using an electrical current. Once the proteins are bound to the nitrocellulose, a specific antibody (see below for a little reminder on what antibodies are) that recognizes the protein of interest is added to the membrane. This antibody binds specifically to the protein of interest, but not to other proteins. The band to which the antibody binds can be determined by tagging the antibody(directly, or indirectly, through another antibody) with a reagent that gives a color reaction. The protocol for the western blot (also called an immunoblot) is given below, and the details of the procedure will be discussed in the lecture.
In this experiment you will use the western blot to examine the differences in the expression of a specific protein at different developmental stages. Recall that although each cell in a multicellular organism has all the instructions for making all the proteins that the organism will need, not all of the proteins are made all of the time. While some proteins are made in certain cells and not others, some proteins are made at some developmental stages and not others. A classic example of proteins that are developmentally regulated are the immunoglobulins (what we commonly call antibodies). In mammals, antibodies are not produced by the developing fetus, or by neonates (newborns). As the immune system develops, the young animal gradually begins to produce its own antibodies. In this experiment, you will compare the serum proteins from different stages of development in cows. First, you will separate the serum proteins from fetal, newborn and adult cows, by using SDS-polyacrylamide gel electrophoresis. Then you will visualize the protein(s) that react with a specific antibody preparation. Note that in this experiment the protein we are studying is, itself, an antibody (confused yet?). We will discuss the rationale of this experiment in greater detail in the lecture.
* Antibody: A somewhat simplistic definition of an antibody is that it is a type of protein, secreted by certain immune cells, that can bind to a foreign molecule that invades the body. Each of us has, circulating in the bloodstream, many different antibodies, specific to different foreign substances that our immune systems may have encountered.
The first half of the experiment is identical to the one that you did last class period, i.e., separating proteins by SDS-PAGE. For this, you will need to set up the gels exactly like the last time, then load the samples provided to you, and run the gels. Once the gels are done, the gel "sandwich" is taken apart and the gel place in the western blot apparatus to transfer the proteins to a membrane. I will demonstrate how to set up the blot. After the blot is done, you will stain the membranes with a reversible stain, Ponceau Red to see what proteins are in the samples. You will finish the experiment up to this point on Wednesday, 5/8. You will then leave the blot in a buffer till the next class period.
Given below are protocols for staining the membrane after the proteins have been transferred on to the nitrocellulose and for visualizing the protein of interest (immunoglobulin G) by using an antibody directed against cow immunoglobulin G.
Protocol
A. Staining the blot
1. Disassemble the blot apparatus, remove the nitrocellulose membrane and place it in 20 mL of protein blot solution in a small Tupperware container.
2. After 5 minutes, pour off the stain and rinse the blot three times with distilled water.
You should see red bands representing the transferred proteins on your membrane.
3. Photograph or photocopy the stained blot and identify the protein bands.
4. Place the blot in 25 mL of gelatin block solution, incubate at 37º C for 10 minutes.
5. Pour off gelatin solution and add a further 25 mL of fresh gelatin solution. Incubate for 10 more minutes at 37º C.
The blots may now be stored for up to a week in the refrigerator. At this point, the red dye should have washed out and the blots should be colorless again.
B. Detection of Immunoglobulin G on the blots:
1. Remove the tray with the blot from the refrigerator and place for 10 minutes in the 37 º C incubator to liquefy the gelatin.
2. Dilute the anti-cow IgG antibody by adding 25 µL of antibody to 5 mL of fresh gelatin solution.
3. Place the blot in the antibody solution and swirl gently to cover the whole membrane with the solution.
4. Place the container with the blot on the rocker at 37º C for 20 minutes.
5. Wash the blot for 3-5 minutes each in 50 mL of the following solutions (add the solution to the blot, place on the rocker for the correct amount of time, for each wash).
-TBS + NP40
-TBS + NP40
-TBS + NP40
-TBS
5. Place 30 mL of the color development solution in a separate container and transfer the blot to it. Rock the tray gently till purple bands appear (5-15 minutes).
6. Rinse the blot in distilled water, air dry it on a paper towel, then place it in your notebook and label the lanes.
7. Compare the results of the protein staining with the red dye and the detection of immunoglobulin on the western blot. What conclusions can you come to?
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