Today we finish one important experiment and prepare for another. We'll split up once again to utilize the tissue culture room -- half of the class will begin by prepping their 96-well plate for the high throughput cell viability analysis on M2D7. You'll use the multichannel pipetting skills that you learned on M2D1.
When you are not in the TC room, you will be finishing your Western blot and obtaining your first protein-level data of the module.
Since you were last in lab, the teaching staff blocked your nitrocellulose membranes with Odyssey Blocking Buffer (OBB) -- a non-mammalian serum based buffer that is proprietary to Licor, Inc (Lincoln, NE) -- and then incubated the membrane with primary antibody at 4C on a shaker for approximately 16 hours.
Recall that the membranes were cut into various pieces so that we can evaluate a few components of the EGFR signaling pathway. As a reminder, shown below is a diagram of how the membrane was cut (dotted lines indicate cut sites).
The diagram roughly shows the molecular weights of our proteins of interest and where they will be located on the membrane when we scan them today. Use the chart below to locate more information about the particular antibodies that were used in the study. An 'X' indicates that a particular antibody was utilized. So, the number and type of antibodies that were used on your membrane reflect which inhibitor you are testing on M2D7.
Today we will use infrared (IR) secondary antibodies to detect the primary antibodies listed in the table above and then scan the Western blots using a specially constructed microscope located in the Lauffenburger lab to determine the phosphorylation of the EGFR netowrk in response to 50 ng/mL (8.4 nM) EGF + increasing amounts of Erlotinib (the conditions that you stimulated your cells with last time).
Laser light path for Licor Odyssey Scanner. Image from Odyssey handbook.
The Licor Odyssey scanner consists of an inverted microscope with two lasers that excite dyes which emit light in the IR range. We will detect our IR-dye conjugated secondary antibodies at wavelengths of 700 and 800 nm. The 700 nm channel will appear red and the 800 nm channel will appear green. Infrared secondary antibodies provide a more flexible detection platform than the traditional Western blot detection methods that rely on colorimetric or chemiluminescent substrates. Unlike the colorimetric or chemiluminescent detection methods, IR dyes do not require a chemical reaction to occur in order for signal to be detected. This means that the output signal increases with time as the colorimetric or chemiluminescent substrate reaction proceeds -- making timing an important variable in traditional Western blot development. We remove that variable from the equation and control when we want to visualize our Western blot simply by controlling the excitation of the dye.
Day Two of Western Blot Analysis -- Secondary Antibody
First we will wash off the primary antibody and then continue with the blot analysis:
- Using a 500 mL glass bottle and the 10x TBS stock on the front bench, make a 500 mL 1x TBS stock using DI water from the lab sink.
- Now add enough Tween 20 to make a 0.1% solution (TBS-T). Tween-20 is located on the front bench. Shake the bottle well to mix.
- Obtain your blots from the front bench. Pour off the antibody solutions into the sink and add enough TBS-T to cover your membranes -- between 10-15 mL should work, but you don't need to measure this out. Keep in mind that the washing steps work by dilution, so it is a balance between adding enough to create a sink for the primary antibody, but not so much that you make a huge mess on the shaker!
- Shake your container for 10 min.
- Repeat for a total of 3 washes. Note: the time and number of washes was determined previously and can depend on your primary antibody!
- During your washes, dilute the secondary antibodies in 5 mL of OBB. They are light sensitive so find them on the front bench and then wrap your 15 mL conical in aluminum foil.
- For GAPDH (Rabbit) -- use the anti-Rabbit IR800 antibody at 1:10,000
- For EGFR (Goat) / p-EGFR (Rabbit) -- use the anti-Goat IR800 at 1:10,000 + anti-Rabbit IR680 at 1:10,000
- For Akt (Mouse) / pAkt (Rabbit) -- use the anti-Rabbit IR800 at 1:10,000 + anti-Mouse IR680 at 1:10,000
- For Erk (Mouse) / pErk (Rabbit) -- use the anti-Rabbit IR800 at 1:10,000 + anti-Mouse IR680 at 1:10,000
- For STAT3 (Mouse) / pSTAT3 (Rabbit) -- use the anti-Rabbit IR800 at 1:10,000 + anti-Mouse IR680 at 1:10,000
- After the last wash, add your secondary antibody and place it on the dark shaker for 30 min.
- Pour off the secondary antibody and wash the membranes 3x, 7 min in TBS-T
- After the last wash, rinse the membranes 1x with 25 mL PBS. Pour off the PBS and keep in another 25mL of PBS until you can scan the membrane.
Imaging the Western Blot
Sample placement for Western blot on Odyssey scanner. Image from Odyssey handbook
The Odyssey scanner is located in the Lauffenburger lab in room 56-378. In groups of two you will go with a member of the teaching staff to scan your blots. The scanner has a variety of settings that control the resolution of the final image and the amount of light that is collected by the microscope objective. You will learn about those settings at the microscope. Be sure to note them in your lab notebook so that you may include them in your Methods section.
Scanning your Western blot is only the first step! You just did a lot of work to obtain what appears to be observational data. However, we can use densitometry to quantify the change in EGFR phosphorylation. Therefore, once you obtain your Western blot images -- which will be .tif files -- use ImageJ (a freeware for biological imaging from the NIH) to perform a densitometric analysis on your bands. A tutorial from NAVBO is attached here for instructions on performing densitometry. You will use this analysis in the FNT assignment for next time and it will help you to limber up your mind for the viability analysis on M2D7. Remember to normalize for total protein content using GAPDH or total Erk signal. Also, in the case of EGFR, you must also take into account the possibility of protein degradation. See the lecture notes, slide 45, for more information.
Setting up the High throughput viability assay
In the tissue culture room you will stimulate a 96-well plate of cells that was seeded for you last night by the teaching staff. The seeding density for this experiment is 31,250 cells/cm2.
Just like on M2D1, you will have a dilution plate and an experimental plate -- this time your experimental plate has cells plated in it!
Your dilution plate will follow this layout:
- E = Erlotinib
- X = Your inhibitor (LY29004, U0126, or Stattic)
- PC = Positive Control (McCoy's + 1% serum + 1% DMSO)
- EGF = Epidermal growth factor
- M = Media (McCoy's + 1% serum + 12.5 ng/mL EGF)
- M+D = Media + 0.001% DMSO
Your experimental plate will follow this layout:
Prepare the dilution plate
Each well in the experimental plate will contain 100 μL of total volume. Half of that volume (50 μL) will contain Erlotinib and the other half (50 μL) will contain inhibitor X. The inhibitors will mix and be diluted 2x when you transfer them from the dilution plate to the experimental plate. Therefore, we will double the highest concentration of each inhibitor when preparing the dilution plate -- for example, the highest concentration of Erlotinib on our experimental plate will be 10 μM, so we will double that to 20 μM on our dilution plate. If this doesn't make sense to you, stop here and ask the teaching staff.
Set-up the dilution plate as follows (use a multichannel pipette and trough where you can):
- To each M well in the dilution plate, add 225 μL of McCoy's media + 1% serum + 12.5 ng/mL EGF.
- To each M+D well in the dilution plate, add 200 μL of McCoy's media + 1% serum + 12.5 ng/mL EGF + 0.001% DMSO.
- To each well marked E20 or X1, add 275 μL of Erlotinib (20μM) or Inhibitor X stock solution, respectively.
- LY294002 stock = 40 μM
- U0126 stock = 20 μM
- Stattic stock = 40 μM
- Starting with the Erlotinib stock solution, use the multichannel pipette with 6 pipette tips attached to move 25 μL from row A to row B by only pressing the pipette plunger to the first stop.
- Mix your solutions by pipetting up and down 4-5 times -- but always stop at the first stop to avoid adding air bubbles!
- Using the same pipette tips, move 25 μL from row B to row C. Repeat your mixing and serial dilutions until row E.
- Do not transfer inhibitor to row F.
- Next, repeat the same protocol diluting the Inhibitor X stock solution across columns 8-11.
- Do not transfer inhibitor to column 12.
- To wells G1 and H1, add 250 μL of PC (Media + 1% DMSO)
- Now, set-up your EGF dilutions. This must be done in eppendorf tubes because the volume is too large.
- Label 5 eppendorf tubes with:
- 50 ng/mL
- 25 ng/mL
- 12.5 ng/mL
- 6.25 ng/mL
- 0 ng/mL
- Add 250 μL of McCoy's media + 1% serum to each eppendorf tube.
- Pick up an aliqout of 500 μL of 100 ng/mL EGF (15.8 nM) from the center bench.
- Serial dilute two-fold (remove 250 μL from the highest concentration and put into the 50 ng/mL tube) and mix well.
- Repeat until you've diluted to 6.25 ng/mL EGF. Do not add EGF to the tube labeled 0 ng/mL.
- Finally, transfer 250 μL of each EGF solution to the appropriate well in the dilution plate.
- We are adding 12.5 ng/mL of EGF to each well with inhibitor so that we can control how much we activate the EGFR signaling pathway. And, as you will likely see from the EGF dose-response curve (in green), using too much EGF will result in cell death by itself! Therefore, a dose of 12.5 ng/mL EGF has been empirically determined to stimulate cell growth in the SKOV3 cells prior to our experiment.
- We will add 1% serum to our media because the cells will die due to lack of serum components after 48 hours and we interested in quantifying the efficacy of the inhibitors, not death due to lack of nutrients. Unfortunately, EGF alone will not sustain these cells.
- To the wells without inhibitor we are adding 0.001% DMSO because, similar to the inhibitor you used in Module 1, our inhibitors are resuspended in DMSO.
Prepare the experimental plate
Finally, let's prepare our experimental
plate. You should read through this protocol and remember what you did on M2D1 -- this is important because the first thing you will do is remove almost all the media from on top of your cells. You don't want to leave them dry for long because they will die. Recall your experimental
plate from M2D1 before moving on.
- Aspirate almost all of the media from the cells in your experimental plate using the vacuum aspirator. You do not need to change aspirating tips or rinse with ethanol between each step. Do this step quickly, but with care not to touch the cells!
- Using the multi-channel pipette with 6 pipette tips attached, add 50 μL from column 12 from your dilution plate to columns 6 and 12 of a new 96-well plate (your experimental plate).
- Note: We deliberately started at the lowest drug concentration (zero!) because that will allow you to re-use the same pipette tips for the next steps.
- Add 50 μL from column 11 of the dilution plate to columns 5 and 11 of the experiment plate.
- Add 50 μL from column 10 of the dilution plate to columns 4 and 10 of the experiment plate.
- Repeat these steps until all of the columns contain increasing Inhibitor X.
- Add 6 new pipette tips to your multi-channel pipetteman.
- Now add the Erlotinib -- begin by pipetting the drug contents (50 μL) of row F from the dilution plate into row F of the experiment plate.
- Note: You will need to perform two pipetting steps to fill all of row F (columns 1-6 and then 7-12).
- Change your pipette tips with each new addition so that you do not contaminate your Erlotinib stock with Inhibitor X
- Continue by adding 50 μL from row E of the dilution plate to row E of the experiment plate.
- Fill the remaining rows with increasing Erlotinib.
- Finally, transfer the media containing the PC and EGF dose-response curve to your experimental plate. You may also use a multi-channel pipette for this step and you do not need to change tips between pipetting steps.
Once you are finished, return your experimental plate to the incubator and aspirate the remaining contents of your dilution plate. Clean up the tissue culture hood and you are finished with tissue culture for the semester!
For Next Time
1. Prepare a figure containing (1) your Western blot images and (2) a densiometric analysis of EGFR and your protein of interest (Akt, Erk, or STAT3). Write a figure caption employing the skills that you learned in Module 1. Refer to lecture notes for an example of how to prepare Western blot figures.
2. Draft the sub-section of your Results that will accompany the figure that you constructed. Here are some things to keep in mind when drafting your Results section:
- Each sub-section should begin with an overview sentence that motivates and introduces the experiment. What did you do and why did you do it?
- State the results of the experiment, minimizing any interpretation of the data (save that for the discussion!)
- Concluding sentences for each paragraph will transition to the next piece of data when possible -- stick to one topic per paragraph, but each sub-section might have a few paragraphs each.
For more guidance in constructing this first draft of your Results section, see the following resources:
Notes for Teaching Faculty
TA notes, mod 2