20.109(F13): Mod 2 Day 6 Transfection of SH2 Domains

Introduction

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.

			Did your team choose to inhibit?
Antibody	Species	Approx. MW	Akt pathway?	Erk pathway?	STAT3 pathway?
EGFR	Goat	150 kDa	X	X	X
tyrosine 1068 pY1068-EGFR	Rabbit	150 kDa	X	X	X
GAPDH	Rabbit	37 kDa	X		X
pS473-Akt	Rabbit	64 kDa	X
total Akt	Mouse	64 kDa	X
pT202/pY204-Erk	Rabbit	42/44 kDa		X
total Erk	Mouse	42/44 kDa		X
pY705-STAT3	Rabbit	75 kDa			X
total STAT3	Mouse	75 kDa			X

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:

1. 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.
2. 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.
3. 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!
4. Shake your container for 10 min.
5. Repeat for a total of 3 washes. Note: the time and number of washes was determined previously and can depend on your primary antibody!
6. 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
7. After the last wash, add your secondary antibody and place it on the dark shaker for 30 min.
8. Pour off the secondary antibody and wash the membranes 3x, 7 min in TBS-T
9. 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 BRET analysis on M2D7.

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 this morning 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:

Here:

• E = Erlotinib
• X = Your inhibitor (LY29004, U0126, or Stattic)
• EGF = Epidermal growth factor

Your experimental plate will follow this layout: File:Experimental plate M2D6.jpg For Next Time 1. Prepare a figure containing your Western blot images and 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. 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: The 20.109 Guidelines for writing up your research Leslie's IMRD Workshop slides Also, you can take a peek at the rubric that will be used to grade your final Mod2 research article. Notes for Teaching Faculty TA notes, mod 2