20.109(F13): Mod 2 Day 4 Phosphotyrosine Western Blot Analysis

Introduction

As we've discussed in lecture, aberrant activation of signaling networks -- such as the EGFR pathway -- contributes to the unchecked growth rate and increased migratory capacity of tumor cells. If we want to learn how to manipulate these networks to our advantage, i.e. to stop cell growth or invasion, we must understand how the network is activated and, better yet, how to inhibit the activation!

How does information flow through an intracellular signaling pathway? Upon binding of ligand, receptor tyrosine kinases (RTKs) facilitate their own tyrosine phosphorylation events. In the case of EGFR, there are six tyrosine autophosphorylation sites on the intracellular tail of the receptor. In addition to those sites, other kinases within the cell, once activated by the signaling pathway, are free to further phosphorylate the receptor. The SH2 (Src Homology 2) domains that you chose from on M2D1 for use in your high-throughput screen are constructed of a conserved amino acid motif that binds with high affinity to phosphorylated tyrosines.

Part 1: Stimulate and lyse cells

Please read Part 1 all the way through before starting the experiment. This part of M2D4 is both time and temperature sensitive. The teaching staff has serum starved your CHO-K1-EGFR-EGFP cells for 4 hrs prior to your arrival. Now you will activate the EGF receptor by perturbing the network and measuring the level of phosphorylation by Western blot.

1. Prepare 6 eppendorf tubes by labeling them with your experimental conditions:
   • No EGF
   • 100 ng/mL
   • 50 ng/mL
   • 25 ng/mL
   • 12.5 ng/mL
   • 6.25 ng/mL
2. Place the eppendorfs on ice.
3. Pick up the following from the front bench ice bucket and put on ice at your bench:
   • 25 mL ice-cold PBS.
   • Lysis buffer
   • Protease Inhibitor
   • Phosphatase Inhibitor
   • Plastic cell scraper
4. Add 10 μL each of protease inhibitor and phosphatase inhibitor to the lysis buffer. Mix well.
5. While one partner obtains the EGF solutions from the front bench, the other partner should remove the 6-well plate from the TC incubator and bring it to the main lab.
   • The EGF dilutions have been prepared in the same buffer that we will use for the BRET assays on M2D7. BRET Buffer = PBS + 1mM Sodium Pyruvate + 1% w/v Glucose.
6. Aspirate the starvation media from each well.
7. Quickly add 1 mL of EGF solution from each eppendorf to one well of the 6-well plate using your P1000 pipette and aiming for the side of the well (avoiding a direct hit to the cells!)
8. Set your timer for 15 min and start it. Leave your plate flat on the benchtop during the incubation.
9. When 30 sec are remaining on the timer, place the plate tilted at an angle in your ice bucket.
10. When your timer goes off, aspirate the EGF from each well and add 1 mL of ice-cold PBS.
11. Aspirate the ice-cold PBS and repeat the wash 1x – make sure to remove ALL of the PBS after this wash.
12. Add 100 μL of lysis buffer across the top of each well, allowing it to run down the well.
13. Collect the cells to the bottom of the well by scraping each well with the cell scraper.
14. In between wells, dip the cell scraper in ethanol and dry with a kimwipe.
15. Add the contents of each well to its respective eppendorf tube.
16. Incubate the eppendorf tubes on ice for 5 min.
17. Spin the tubes at max speed in the cold room centrifuge for 10 min to pellet insoluble material. Bring your eppendorf tubes to the TA who will spin them for you.
18. Meanwhile, label a second set of eppendorf tubes as in Step 1 and chill.
19. Transfer the supernatant to the new set of eppendorf tubes and keep on ice – be careful not to disturb the pellet at the bottom!

Part 2: Measure protein concentration

You will now measure the total protein concentration in our cell lysate to determine the volume required to evaluate EGFR phosphorylation and total expression by Western blot. We are using the Precision Red Advanced Protein Assay from Cytoskeleton.

• Add 10 μL of cell lysate to duplicate wells in the 96-well plate on the front bench following this layout:

1	2	3	4	5	6	7	8	9	10	11	12
Red 0	Red 0	Red 100	Red 100	Red 50	Red 50	Red 25	Red 25	Red 12.5	Red 12.5	Red 6.25	Red 6.25
Orange 0	Orange 0	Orange 100	Orange 100	Orange 50	Orange 50	Orange 25	Orange 25	Orange 12.5	Orange 12.5	Orange 6.25	Orange 6.25
Yellow 0	Yellow 0	Yellow 100	Yellow 100	Yellow 50	Yellow 50	Yellow 25	Yellow 25	Yellow 12.5	Yellow 12.5	Yellow 6.25	Yellow 6.25
Green 0	Green 0	Green 100	Green 100	Green 50	Green 50	Green 25	Green 25	Green 12.5	Green 12.5	Green 6.25	Green 6.25
Blue 0	Blue 0	Blue 100	Blue 100	Blue 50	Blue 50	Blue 25	Blue 25	Blue 12.5	Blue 12.5	Blue 6.25	Blue 6.25
Pink 0	Pink 0	Pink 100	Pink 100	Pink 50	Pink 50	Pink 25	Pink 25	Pink 12.5	Pink 12.5	Pink 6.25	Pink 6.25
Purple 0	Purple 0	Purple 100	Purple 100	Purple 50	Purple 50	Purple 25	Purple 25	Purple 12.5	Purple 12.5	Purple 6.25	Purple 6.25
Platinum 0	Platinum 0	Platinum 100	Platinum 100	Platinum 50	Platinum 50	Platinum 25	Platinum 25	Platinum 12.5	Platinum 12.5	Platinum 6.25	Platinum 6.25
White 0	White 0	White 100	White 100	White 50	White 50	White 25	White 25	White 12.5	White 12.5	White 6.25	White 6.25

• Once all samples have been added to the plate we will add 290 μL of Precision Red reagent to each well using a multichannel pipetman.
• Measure the OD₆₀₀ using the 96-well plate reader located in 56-421 after a one minute incubation. (Kim will do this for you!)
• The OD₆₀₀ values will be projected at the front of the room -- calculate the total protein concentration using the following relationship:
  

(1.00 OD₆₀₀ = 1.25 μg/mL of total protein) * dilution factor

• You may use the table below to calculate the volume required to add 20 μg of total protein in each well of your SDS-PAGE gel or setup your own Excel spreadsheet to do the calculations automatically.

Sample Name	OD600 1	OD600 2	Protein 1 (μg/mL)	Protein 2 (μg/mL)	Avg Protein (μg/mL)	Avg Protein (μg/μL)	Vol for 20 μg
100
50
25
12.5
6.25
0

Fill out this table (or your Excel spreadsheet) before moving to Part 3.

Part 3: Protein Gel (SDS-PAGE)

Each group will run a lane of molecular weight markers, a lane with a positive control for the Western (e.g. an aliquot of purified His6-EnvZ), a lane with the wild type light sensor, and two lanes of mutants from your library screen. Two teams will share one gel.

1. Retrieve the bacterial cultures carrying the wild type or mutant light sensors that have been grown.
2. To compare intensities between lanes on the protein gel it's necessary that equal numbers of cells be loaded into each well. You'll assess the number of cells in each sample by making a 1:10 dilution of the three strains in Z-Buffer and use the spectrophotometer to measure the density of the samples at a wavelength of 600 nm. This measurement tells you something about the number of cells in a millileter of liquid. For example a reading of 0.7 says the sample has 0.7 OD units of cells / ml.
3. Calculate the volume of your cells needed to give 2 OD. Thinking again about a sample that reads 0.7 OD: if you wanted to collect the number of cells equivalent to 2 OD unit, then you would have to collect 2/0.7 = 2.85 ml of that sample to get 2 OD's worth of cells. Heads up: don't forget that your spectrophotometric reading is for a 1:10 dilution of the original (undiluted) samples, so if you go back to the overnight cultures you'll have to take that dilution factor into account.
4. Move the calculated volume of cells to well-labeled eppendorf tubes, and spin the tubes in a microfuge for 1 minute to pellet the bacteria. Be sure that each eppendorf is balanced in the microfuge with an opposing eppendorf containing the same volume. You can collect greater than 1.5 ml of cells by dividing the total volume into smaller aliquots that do fit in the eppendorfs, harvesting the cells, removing the supernatants from the pellets and then adding more culture volume to the same eppendorf tube.
5. Resuspend each pellet in 100 ul of "EasyLyse" protein extraction solution (a commercial product from a company called Epicentre). Incubate the solutions at room temperature for 5 minutes, then pellet the debris by spinning the tubes in the microfuge (full speed) for 2 minutes. The supernatant is the lysate that you will run on your protein gel.
6. Mix 30 ul of the bacterial lysates with 30 ul of 2X sample buffer. Sample Buffer contains glycerol to help your samples sink into the wells of the gel, SDS to coat amino acids with negative charge, BME to reduce disulfide bonds, and bromophenol blue to track the migration of the smallest proteins through the gel. Wear gloves when using sample buffer or your hands will get blue and smelly.
7. Prepare your positive control H6-EnvZ protein tube by moving 50 ul of the sample to an eppendorf tube.
8. Put lid locks on the eppendorf tubes and boil for 5 minutes.
9. Put on gloves. Load the indicated volumes of each sample onto your acrylamide gel in the order below. Once you have loaded a sample from one tube, move it to a different row in your eppendorf tube rack. This will help you keep track of which samples you have loaded.

Lane	Sample	Volume to load
1	"Kaleidoscope" protein molecular weight standards	10 ul
2	H6-EnvZ positive control protein	40 ul
3	wild type light sensor	40 ul
4	mutant candidate 1	40 ul
5	mutant candidate 2	40 ul
6	"Kaleidoscope" protein molecular weight standards	10 ul
7	H6-EnvZ positive control protein	40 ul
8	wild type light sensor	40 ul
9	mutant candidate 1	40 ul
10	mutant candidate 2	40 ul

10. Once all the samples are loaded, turn on the power and run the gel at 200 V. The molecular weight standards are pre-stained and will separate as the gel runs. The gel should take approximately one hour to run. During that hour, you should work on part two of today's protocol.
11. Wearing gloves, disassemble the electrophoresis chamber.
12. Blot the gel to nitrocellulose as follows:

• Place the gray side of the transfer cassette in a tupperware container which is half full of transfer buffer. The transfer cassette is color-coded so the gray side should end up facing the cathode (black electrode) and the clear side facing the anode (red).
• Place a ScotchBrite pad on the gray side of the cassette.
• Place 1 piece of filter paper on top of the ScotchBrite pad.
• Place your gel on top of the filter paper.
• Place a piece of nitrocellulose filter on top of the gel. The nitrocellulose filter is white and can be found between the blue protective paper sheets. Wear gloves when handling the nitrocellulose to avoid transferring proteins from your fingers to the filter.
• Gently but thoroughly press out any air bubbles caught between the gel and the nitrocellulose.
• Place another piece of filter paper on top of the nitrocellulose.
• Place a second ScotchBrite pad on top of the filter paper.
• Close the cassette then push the clasp down and slide it along the top to hold it shut.
• Place the transfer cassette into the blotting tank so that the clear side faces the red pole and the gray side faces the black pole.
  

13. Two blots can be run in each tank. When both are in place, insert the ice compartment into the tank. Fill the tank with buffer. Be sure the stir bar is able to circulate the buffer. Connect the power supply and transfer at 100 V for one hour. You can use this time to complete the other parts of today's protocol.
14. After an hour, turn off the current, disconnect the tank from the power supply and remove the holders. Retrieve the nitrocellulose filter and confirm that the pre-stained markers have transferred from the gel to the blot. Move the blot to blocking buffer (TBS-T +5% milk) and store it in the refrigerator until next time.

Notes for Teaching Faculty

TA notes, mod 2