BISC220/S12: Mod 2 Lab 7

Background Information
Lab 5: Phenotypes of Secretory Mutants
Lab 6: Analyzing Secretion Defects by Western Blotting
Lab 7: Probing and Detecting the Western Blot
Media Recipes

Probing and Developing the Western Blot

Primary and Secondary Antibodies
A variety of procedures have been developed to visualize proteins on Western blots. Most often, an antibody made against the protein of interest (primary antibody) is used to recognize the protein. A secondary antibody that binds to the primary antibody is then used to visualize the spots on the nitrocellulose where the primary antibody has bound. The secondary antibody is conjugated to an enzyme that produces a visible product. As discussed in lecture, the two-antibody scheme has two main advantages over using a single antibody:

1. The signal is amplified because more than one secondary antibody molecule can bind to each primary antibody;
2. There is no need to generate an enzyme-conjugated version of each primary antibody.

For our Western blot, we will use a primary antibody that recognizes pre-pro-α-factor to identify the various forms of α-factor that accumulated in the sec18 and sec61 yeast strains that were shifted to the non-permissive temperature (37°C). Remember that pre-pro-α-factor is the entire polypeptide that is synthesized by the ribosome, including the ER signal sequence and other sequences that are removed by proteolysis to generate the final, secreted form of α-factor. The anti-pre-pro-α-factor antibody was generated in a rabbit. We will therefore use a goat anti-rabbit antibody as our secondary antibody. This antibody is linked to the enzyme horseradish peroxidase (HRP), which will be used to visualize the immunoreactive proteins by a method called enhanced chemiluminescence.

Enhanced Chemiluminescence (ECL)
Luminescence refers to energy that is dissipated in the form of light when some molecules transition from an excited state to their ground state. When the excitation of the molecule is caused by a chemical reaction, it is referred to as chemiluminescence. Examples of chemiluminescence are the light produced by fireflies and the glow of Halloween light sticks. In the chemical reaction that will be used to detect the HRP-conjugated goat-anti-rabbit antibodies on our Western blots, the light releasing molecule is a compound called luminol. Luminol is commonly used in crime scene investigations to visualize traces of blood. Luminol is mixed with hydrogen peroxide (H₂O₂) and is applied to the surface that is being investigated. If traces of blood are present, the iron in the blood acts as a catalyst in the oxidation of luminol, which is accompanied by blue-green luminescence. In our case, the HRP linked to the secondary antibody will catalyze the same reaction, and we will detect the light produced by exposing our blots to blue light-sensitive film. Figure 1 below provides a summary of the detection method. Enhanced chemiluminescence refers to the fact that, in addition to the H₂O₂ and the luminol, the developing solutions we will use also contain some additional chemicals (enhancers) that increase the intensity of the light produced in the reaction.

Figure 1. A summary of the ECL Western blot detection method. The secondary antibody is conjugated to the enzyme horseradish peroxidase (HRP). When this enzyme is activated by hydrogen peroxide (H₂O₂) and provided with the substrate luminol, the luminol becomes oxidized and releases light, which can be detected on film. When the film is developed, the spots on the blot where the antibodies have bound will be black. The enhancers are additional chemicals that amplify the light produced from the reaction.

ECL Detection Protocol

Microsoft Word File: Media:ECL Detection PROTOCOL.doc

1. Each blot has been incubating in 10 ml of blocking buffer for a hour on an orbital shaker. (The blocking buffer contains 5% milk in phosphate buffered saline with 0.25% Tween, a detergent. The proteins in the milk bind non-specifically to the nitrocellulose, “blocking” sites where the primary antibodies might bind non-specifically.)
2. Pour off the blocking buffer into the sink (try to leave as little volume as possible so the primary antibody will not be further diluted). Add the primary antibody (rabbit anti-pre-pro-α-factor), which has already been diluted 1:1000 in 10 ml of blocking buffer. Incubate on the rocker for 1 hour. *Note: you will need to save the primary antibody for use by other lab groups. Do not discard it after the incubation is finished.
3. Return the primary antibody solution to the conical centrifuge tube and give it back to your instructor (try to recover as much volume as possible). Pour PBST wash buffer onto the nitrocellulose so that it is submerged by approximately 0.5 cm of liquid. Incubate 10 min. on the shaker.
4. Pour off the wash buffer into the sink. Add fresh wash buffer and incubate another 10 min. on the shaker.
5. Repeat Step 4 once more (a total of 3 10-min. washes).
6. Add the HRP-conjugated goat anti-rabbit secondary antibody that has been diluted 1:2500 in 10 ml blocking buffer (i.e. add 4 µl of antibody to 10 ml of blocking buffer). Incubate 30 min. on the shaker.
7. Pour off the secondary antibody solution into the sink (you do not need to save this).
8. Wash three times with PBST (10 min. each wash) as you did after the primary antibody incubation. After the last wash, remove all residual wash buffer by tilting the blot container onto a paper towel. *Do not proceed with the following steps until your instructor is available to help you with the film exposure and developing. Leave your blot in the final batch of wash buffer until that time. The following directions for performing the ECL may be different depending on which manufacturer’s kit we use. Check with your instructor to see if the following step (ratio of reagents 1 &2) needs to be modified before proceeding.
9. Pipet 2 ml of ECL detection reagent 1 into a 15-ml conical tube and add 2 ml of reagent 2 and invert to mix. Be sure to use a clean pipet for each detection reagent — it is important for the two solutions not to mix until just before you use them! Make sure that you record all the kit information in your lab notebook as the reagents are proprietary. You will need to include manufacturer information for using this kit in your M&M section since you don’t know the ingredients and concentrations for these reagents.
10. Pour the mixed ECL developing solution onto your blot. Make sure all the membrane is in contact with the liquid by rocking the container slightly back and forth by hand for 1 min. Discard the ECL reagents in the sink.
11. Working quickly but carefully, use forceps to place your blot onto a piece of paper towel. Using another piece of paper towel, gently blot the nitrocellulose to remove excess liquid.
12. Use forceps to place the membrane blot—protein side up—between the two plastic sheets of a page protector that has been taped into an autoradiography cassette.
13. With your instructor, take the cassette into the darkroom. Hold the open cassette up to the light for about 20 sec. to activate the glow-in-the-dark StratageneTM logo marker. (This will help in aligning your developed film with the MW markers on the blot.)
14. Flip the switch to turn off the normal light and turn on the safe light. (Also, make sure the computer monitor in the room is turned off.) Open the film container and remove one piece of film. Be sure you actually get a piece of film and not one of the thin paper sheets that divide the films. Place the film onto the blot and close the cover of the cassette (be careful not to move the film once it has been placed on the blot). Wait ~1 min. to 90 sec., then open the cassette and remove the film.

Data Interpretation:

1. Estimate the molecular weights of all the bands on your developed film. Just approximate based on the MW markers—you do not need to construct a standard curve as you did for the GelCode Blue-stained gel in the β-gal lab series.
2. What are the dark bands that are present in each sample and what do they represent? What are the likely reasons for the differences in their sizes? Use your knowledge of the specific defects of the sec18 and sec61 strains to help you interpret your data. Also consider what it means if no α-factor signal is present for some of the samples.

SDS PAGE Protocol

Microsoft Word File: Media:SDS PAGE Protocol.doc

1. Label your six tubes of yeast cells with your initials and make sure the cells are thawed. To each tube of cells, add 200 µl of SDS sample buffer (10% SDS, 25% glycerol, 250mM Tris pH6.8, 500mM, DTT, 0.5% BPB) and “1 scoop” of glass beads. The sample buffer contains SDS and a reducing agent to ensure that the proteins are fully denatured. You will find six microfuge tubes, each containing 1 scoops of glass beads, at your bench so you can just pour the beads from these tubes into the screw-capped tubes that contain your yeast cells. The beads are to insure that the tough yeast cell wall is broken in step 2. Your instructor will prepare a tube of negative control Mat a wild type yeast cells by adding 600 µl of sample buffer and 3 scoops of glass beads and processing as in step 2 & 3. These cells will be available at the instructor’s bench for everyone to use.
2. Make sure the caps are tightly closed. Process the samples at room temperature in the FastPrep bead beater for 20 seconds (speed = 6.0). Your instructor will assist you in loading and unloading the samples from the machine. Yeast cells have a tough cell wall, so the glass beads are necessary to lyse the cells.
3. Centrifuge 10 minutes at maximum speed in a microcentrifuge.
4. Label 7 microfuge tubes according to the chart below (Lanes 2-8). Transfer 75 µl of each lysate you prepared and 50 µl of the Mat a yeast lysate that do not express alpha factor (negative control), found on the instructor’s bench, to the correct labeled microcentrifuge tubes, taking care to avoid the beads and cellular debris at the bottom of the lysis tubes.
5. Boil all 7 samples for 3 minutes at 100°C in a heating block. Place a plastic cap holder on each tube to prevent it from popping during heating and spraying your sample everywhere. When you take your samples out of the heating block, carefully vent each tube by opening the caps very slightly (pointing awayItalic text from you) to allow the vapor to escape. Some liquid may spurt out—you can just wipe this off with a KimWipe™.
6. The gels you will be using are 18% Tris-HCl polyacrylamide, which will slow the migration and help us visualize small forms of alpha factor. Each group will run one gel, but two groups can use a single electrophoresis apparatus. When you pipet your samples to load on the gel, draw from the top of the liquid, trying to avoid sucking up any stray glass beads. Load your samples as shown below.
7. Run your gel for ~35 min. at 200 volts.

Gel Loading:
Lane 1 — 20 µl of pre-stained MW standards (do not need to be boiled)
Lane 2 — 20 µl of SEC+, 25°C lysate
Lane 3 — 20 µl of SEC+, 37°C lysate
Lane 4 — 20 µl of sec18, 25°C lysate
Lane 5 — 20 µl of sec18, 37°C lysate
Lane 6 — 20 µl of sec61, 25°C lysate
Lane 7 — 20 µl of sec61, 37°C lysate
Lane 8 — 20 µl of MATa lysate

Finishing the SDS-PAGE & Starting the Anti-pp-αF Western blot Turn the power source controlling your gel electrophoresis to OFF. Remove your gel from the apparatus and separate the plates. Carefully cut off the wells at the top of the gel with a razor blade. Be careful not to tear the gel. Take it over to the blotting apparatus.

The holder for the blotting apparatus is color coded so the black side should end up facing the cathode (black electrode) and the clear side facing the anode (red). The sandwich of choice, therefore, will be made in the following manner, keeping all parts wet in the blotting buffer in the plastic container provided at your bench:

1. Wear gloves when handling the nitrocellulose membranes. The membrane is white, and will be given to you sandwiched between two pieces of blue protective paper. Remove the top piece of blue paper. In pencil, label the top left corner of your piece of nitrocellulose (NC) with your initials. Be careful not to tear the membrane with the pencil tip. Pour some blotting buffer into your small plastic container. Immerse the NC in blotting buffer so that it is wet evenly.
2. Place the blotting holder black side down in another plastic container after filling the container half full with blotting buffer.
3. Open the blotting holder, wet one of the sponges with blotting buffer and place it on the black side of the holder.
4. Wet 2 pieces of 3 mm paper and place them on top of the sponge.
5. Wet your gel with blotting buffer in the small plastic container and place the GEL on top of the 3 mm paper. To make the left-most lane (Lane 1) of the gel come out on the left side of the blot, orient your gel so that Lane 1 (containing the stained MW ladder) is on the right side of the blot sandwich.
6. Place your wet NC on top of the gel.
7. Add 2 more pieces of moistened 3 mm paper on top of the NC. Use a broken plastic pipet like a rolling pin to gently “roll” out any air bubbles out from between the gel and the NC.
8. Place a second moistened sponge on top of the 3 mm paper.
9. Close the clear side of the holder, pushing the clasp down and along the top of the holder.
10. Place the holder into the blotting tank so that the clear side faces the red pole and the black side faces the black pole.
11. Two blots can be run in each tank. Place a frozen ice compartment into the tank. Fill the tanks so the buffer is up to the top of the gel. Connect the top of the tank tightly. Connect the power supply and run the blots at 100 volts for one hour. Be sure the stir bar is free to stir to keep the buffer cold. Watch the current. It should read 0.15-0.20 amps during the run.
12. After an hour, disconnect the power and remove the holders. Remove the NC, rinse it with distilled water, and stain it with Ponceau S protein stain to see if the proteins were transferred properly to your membrane. Pour the 25ml provided of PonceauS onto your NM, placed in the small plastic container and rock back and forth for 15-30 sec. Pour stain back into 50ml conical tube (DO NOT DISCARD!). Rinse off the protein stain by pouring distilled water onto stained blot, rock, and discard water into sink. Look for approximately equal protein bands in all lanes loaded with cell lysates and look for presence of MW marker bands. Once you have determined that your transfer worked and that all lanes have approximately the same amount of protein, allow the membrane to dry a bit, then wrap it in plastic. Label it with your name and lab section. Store the NM in the refrigerator in a container provided by your instructor until the next lab.

Protocol: Finishing the Yeast Spotting Experiment

Microsoft Word File: Media:RESULTS for yeast secretory pathway REV 2010.doc

Scoring the results of the growth phenotype assay:
DO NOT refer to your table of predictions until you have scored your results.

Score growth in the results table according to the following relative scale:
++ , + , +/- , or – (NG)

Consider a spot of growth that is confluent (fills circle completely) as ++.

Rate growth as + if there are many or a few small visible colonies, but growth over the entire spot is not uniform.

Growth can be scored as +/- if there are tiny colonies that are clearly different from the film of cells left from the spotting procedure.

Be very careful not to consider the film of dead yeast cells left from the spotting procedure as growth. Ask your instructor for help if you are not sure how to make this distinction.

After you have recorded your results, compare your results to your predictions. Do they agree? If not, think about why you might see growth that you did not expect or vice versa. Which strains are “controls” and for what do the controls test? Temperature sensitive mutants have variable sensitivity and some strains may be more or less temperature sensitive than expected. Think about which nutrient genes are present or lacking in the plasmids pRSB204, pRSB203 and in the vector plasmid YEp24. Consider whether the gene product is a fusion protein and where in the cell that fusion protein is directed. Now think about how the proteins encoded by the plasmid interact with the SEC gene defects found in the yeast mutants. What differences do you see when comparing the 9 strains’ growth pattern in the same conditions (on the same plate) and what differences do you see when you compare each strain’s growth at different temperatures and on different media?

Now look at your results vs. your expectations. If you have unexpected growth (or no growth) where you expected to see colonies, try to come up with possible explanations for discrepancies. You may have time to begin this analysis while your proteins separate or during the transfer phase of the Western blot. Resolving the growth patterns with expectations may be a challenging exercise. Feel free to discuss your thoughts with your partner or others in the class, but make sure any work that is submitted for grading is entirely your own.