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

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==Notes for Teaching Faculty==
==Notes for Teaching Faculty==
[[20.109(F13): TA notes for module 2| TA notes, mod 2]]
[[20.109(F13): TA notes for module 2| TA notes, mod 2]]
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==Navigation Links==
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Revision as of 15:54, 30 October 2013

20.109(F13): Laboratory Fundamentals of Biological Engineering

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Contents

Introduction

Low throughput analysis -- semi-quantification of phosphorylation for Systems Biology studies

How does one define 'Systems Biology'? One definition is provided by the MIT Integrative Cancer Biology Program:

Systems biology strives to describe the extreme multivariate nature of cellular systems using statistical and mathematical techniques, ultimately predicting the response of cells/tissues/organisms to normal and pathological perturbations. To achieve predictive models, systems biology demands integration of disparate data; genomic, proteomic, metabolomic, or epigenetic data alone is not enough to fully describe organismal behaviors. Therefore, systems biology is necessarily a multidisciplinary venture requiring significant collaboration and melding of experimentation and computation.

Phew, that is a mouth full. Let's boil that down to two key points: Systems biology depends on (1) measuring many parameters and (2) using mathematical relationships to distill those parameters down to the ones that are most important. To satisfy these key points we need an informative experimental system that is amenable to a great number of perturbations. For the purpose of 20.109, 'perturbation' refers to the cell stimulus or intracellular signaling pathway inhibitor that we are employing in this module. However, in a greater context, perturbation could be cell culture media formulation, temperature and oxygen conditions within the TC incubator, elasticity of cell culture surface, 2D vs. 3D culture environment, etc.

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. Tyrosine phosphorylation of EGFR results in recruitment and activation of signaling proteins, kicking off the canonical signaling cascades that we are targeting on M2D6/7; PI3K/Akt, Ras/Erk, and STAT3.

To determine how treatment with Erlotinib affects phosphorylation of the components in our system of interest, every group will monitor the phosphorylation of EGFR at tyrosine 1068 using a rabbit monoclonal antibody from Cell Signaling Technologies, Inc. Depending on what inhibitor you have chosen to evaluate on M2D6/7, you will either detect the phosphorylation of:

General Western blot information

For Western analysis, a high quality antibody can have a relatively low affinity for its target protein. This is because the target is localized and concentrated on a blot, allowing the antibody to bind using both antibody “arms” thereby strengthening the association. Even an antibody that is loosely bound to the blot under these circumstances may dissociate then re-associate quickly since the local concentration of the target protein is high. The lower limit for protein detection is approximately 1 ng/lane, a value that varies with the size of the protein to be detected and the Western blotting apparatus that is used. For most acrylamide gels, the protein capacity for each lane is usually 100 to 200 ug (that would be 20 ul of a 5-10 ug/ul protein preparation). Thus 1 ng represents a protein that is approximately 0.001-0.002% of the total cellular protein (1 ng out of 100,000-200,000 ng). Obviously proteins that make up a more significant fraction of the total protein population will be easier to detect.

Many species can be used to raise antibodies. Most commonly mice, rabbits, and goats are immunized, but other animals like sheep, chickens, rats and even humans can be used. The protein used to raise an antibody is called the antigen and the portion of the antigen that is recognized by an antibody is called the epitope. Each antibody can recognize only a small portion of its antigen, typically 5 to 6 amino acids. Some antibodies are monoclonal, or more appropriately “monospecific,” and recognize one epitope, while other antibodies, called polyclonal antibodies, are in fact antibody pools that recognize multiple epitopes.

generating polyclonal antibodies

To raise polyclonal antibodies, the antigen of interest is first purified and then injected into an animal. To elicit and enhance the animal’s immunogenic response, the antigen is often injected multiple times over several weeks in the presence of an immune-boosting compound called adjuvant. After some time, usually 4 to 8 weeks, samples of the animal’s blood are collected and the cellular fraction is removed by centrifugation. What is left, called the serum, can then be tested in the lab for the presence of specific antibodies. Even the very best antisera have no more than 10% of their antibodies directed against a particular antigen. The quality of any antiserum is judged by its purity (that it has few other antibodies), its specificity (that it recognizes the antigen and not other spurious proteins) and its concentration (sometimes called its titer). Animals with strong responses to an antigen can be boosted with the antigen and then bled many times, so large volumes of antisera can be produced. However animals have limited life-spans and even the largest volumes of antiserum will eventually run out, requiring a new animal for immunization. The purity, specificity and titer of the new antiserum will likely differ from that of the first batch. High titer antisera against bacterial and viral proteins can be particularly precious since these antibodies are difficult to raise; most animals have seen these immunogens before and therefore don’t mount a major immune response when immunized. Antibodies against toxic proteins are also challenging to produce if they make the animals sick.

generating monoclonal antibodies

Monoclonal antibodies overcome many limitations of polyclonal pools in that they are specific to a particular epitope and can be produced in unlimited quantities. However, more time is required to establish these antibody-producing cells, called hybridomas, and it is a more expensive endeavor. Antibody-secreting cells are first isolated from an immunized animal, usually a mouse, and then fused with an immortalized cell line such as a myeloma. The fusion can be accomplished by incubating the cells with polyethylene glycol (antifreeze), which facilitates the joining of the plasma membranes of the two cell types. A fused cell with two nuclei can be resolved into a stable hybridoma after mitosis. The unfused antibody-secreting cells have a limited lifespan and so die out of the hybridoma population, but the myelomas must be removed with some selection against the unfused cells. Production of stable hybridomas is tedious and difficult but often worth the effort since monoclonal antibodies can recognize covalently-modified epitopes specifically. These are invaluable for experimentally distinguishing the phosphorylated or glycosylated forms of an antigen from the unmodified forms.

Making antibodies is big business since they can be useful therapeutics. Whereas the 2002 market for monoclonal therapeutic antibodies was estimated at almost $300 million, sales grew to $43 billion in 2010 and are predicted to reach nearly $58 billion in 2016 link. Successful antibody treatments, however, require clever engineering discoveries to “humanize” antibodies raised in other animals, as well as speedier development, well-protected patents, improvements in drug-delivery methods and cost efficient production of the therapeutics.

Protocol

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 SKOV3 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.

All of your cells will be stimulated with 50 ng/mL (~ 8.4 nM) EGF -- the same amount of EGF that we simulated in our ODE models on M2D1. To determine the effect of Erlotinib on the phorphorylation of EGFR and downstream proteins (Erk, Akt, and STAT3), we will perform a dose-inhibition study by increasing the amount of Erlotinib in each well of the 6-well plate.

  1. Prepare 6 eppendorf tubes by labeling them with your experimental conditions:
    • No Erlotinib (easy label = No Erl)
    • 10 μM
    • 1 μM
    • 0.1 μM
    • 0.01 μM
    • 0.001 μM
  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 Erlotinib 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 Erlotinib dilutions have been prepared in serum free McCoys 5A media.
  6. Aspirate the starvation media from each well.
  7. Quickly add 1 mL of Erlotinib 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 Erlotinib from each well and add ice-cold PBS by pouring on the cells.
  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 10 Red 10 Red 1 Red 1 Red 0.1 Red 0.1 Red 0.01 Red 0.01 Red 0.001 Red 0.001
Orange 0 Orange 0 Orange 10 Orange 10 Orange 1 Orange 1 Orange 0.1 Orange 0.1 Orange 0.01 Orange 0.01 Orange 0.001 Orange 0.001
Yellow 0 Yellow 0 Yellow 10 Yellow 10 Yellow 1 Yellow 1 Yellow 0.1 Yellow 0.1 Yellow 0.01 Yellow 0.01 Yellow 0.001 Yellow 0.001
Green 0 Green 0 Green 10 Green 10 Green 1 Green 1 Green 0.1 Green 0.1 Green 0.01 Green 0.01 Green 0.001 Green 0.001
Blue 0 Blue 0 Blue 10 Blue 10 Blue 1 Blue 1 Blue 0.1 Blue 0.1 Blue 0.01 Blue 0.01 Blue 0.001 Blue 0.001
Pink 0 Pink 0 Pink 10 Pink 10 Pink 1 Pink 1 Pink 0.1 Pink 0.1 Pink 0.01 Pink 0.01 Pink 0.001 Pink 0.001
Purple 0 Purple 0 Purple 10 Purple 10 Purple 1 Purple 1 Purple 0.1 Purple 0.1 Purple 0.01 Purple 0.01 Purple 0.001 Purple 0.001
Platinum 0 Platinum 0 Platinum 10 Platinum 10 Platinum 1 Platinum 1 Platinum 0.1 Platinum 0.1 Platinum 0.01 Platinum 0.01 Platinum 0.001 Platinum 0.001
White 0 White 0 White 10 White 10 White 1 White 1 White 0.1 White 0.1 White 0.01 White 0.01 White 0.001 White 0.001


  • 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 A600 using the 96-well plate reader located in 56-421 after a one minute incubation. (Kim will do this for you!)
  • The A600 values will be projected at the front of the room -- calculate the total protein concentration using the following relationship:

If A600 = 1.00, the concentration of total protein is 125 μg/mL.

  • Don't forget your dilution factor of 30!
  • You may use the table below to calculate the volume required to add 30 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 A600 1 A600 2 Protein 1 (μg/mL) Protein 2 (μg/mL) Avg Protein (μg/μL) Vol for 30 μg Vol of Water to get to 15 μL Vol of 6x sample buffer
10 3 μL
1 3 μL
0.1 3 μL
0.01 3 μL
0.001 3 μL
0 3 μL
  • Finally, calculate the amount of water for a total volume of 15 μL
  • If you can't fit 30 μg of total protein within your 15 μL, redo the calculations for 20 μg and make a note of this in your notebook.

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

Part 3: Protein Gel (SDS-PAGE) & Protein Transfer

Two teams will share one gel.

  1. Set up well-labeled eppendorf tubes with the volume of reagents calculated in the table above.
  2. Put lid locks on the eppendorf tubes and boil for 5 minutes.
  3. Do not throw away the remainder of your cell lysate! We will store this for you at -80C in case another Western blot needs to be completed.
  4. Load 15 μL 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 0 15 μL
3 0.001 15 μL
4 0.01 15 μL
5 0.1 15 μL
6 1 15 μL
7 10 15 μL
8 No Sample 0 ul
9 "Kaleidoscope" protein molecular weight standards 10 μL
10 0 15 μL
11 0.001 15 μL
12 0.01 15 μL
13 0.1 15 μL
14 1 15 μL
15 10 15 μL

5. Once all the samples are loaded, turn on the power and run the gel at 125 V. The molecular weight standards are pre-stained and will separate as the gel runs. The gel should take approximately 45 minutes to run.
6. Wearing gloves, disassemble the electrophoresis chamber.
7. 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 2 pieces 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 2 pieces 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.

8. 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. Connect the power supply and transfer at 100 V for one hour.
9. 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 Odyssey blocking buffer and store it in the refrigerator until next time. The teaching staff will do step #9 for you.

10. Before moving the blot to buffer, the teaching staff will cut the blot using the following map so that you may visualize all of your primary antibody targets:

Reagents

  1. 100X Protease Inhibitor cocktail (Boston Bioproducts)
    • AEBSF
    • Aprotinin
    • E-64 Besstain Leupeptin
    • EDTA
  2. 100x Phosphatase Inhibitor cocktail (Boston Bioproducts)
    • This cocktail will inhibit acid, alkaline, and tyrosine phosphatases
    • Imidazole
    • Sodium fluoride
    • Sodium molybdate
    • Sodium orthovanadate
    • Sodium pyrophosphate tartrate
  3. RIPA Lysis Buffer (Boston Bioproducts)
    • 50 mM Tris-HCL, pH 7.4
    • 150 mM NaCL
    • 1% NP-40
    • 0.5% Sodium deoxycholate
    • 0.1% SDS
  4. 6x Reducing Laemlli Sample Buffer (Boston Bioproducts)
    • 375 mM Tris HCL, pH 6.8
    • 9% SDS
    • 50% Glycerol
    • 9% Betamercaptoethanol
    • 0.03% Bromophenol blue
  5. Tris-Glycine-SDS Running Buffer (Boston Bioproducts)
    • 25 mM Tris base, pH 8.3
    • 192 mM Glycine
    • 0.1% SDS
  6. Transfer Buffer
    • 25 mM Tris
    • 192 mM Glycine
    • 20% v/v Methanol
  7. Mini-PROTEAN TGX gels (Bio Rad)
    • 4-20% gradient polyacrylamide
    • 15-well
  8. Licor Odyssey Blocking Buffer (Licor)
    • Proprietary formulation with non-mammalian serum
  9. Primary Antibodies
    • Anti-EGFR, raised in goat (Santa Cruz Biotechnology)
    • Anti-pY1068-EGFR, raised in rabbit (Cell Signaling Technologies)
    • Anti-Akt, raised in mouse (Cell Signaling Technologies)
    • Anti-pS473-Akt, raised in rabbit (Cell Signaling Technologies)
    • Anti-ERK, raised in mouse (Cell Signaling Technologies)
    • Anti-pT202/Y204-ERK, raised in rabbit (Cell Signaling Technologies)
    • Anti-STAT3, raised in mouse (Cell Signaling Technologies)
    • Anti-pY705-STAT3, raised in rabbit (Cell Signaling Technologies)
    • Anti-GAPDH, raised in rabbit (Santa Cruz Biotechnology)
  10. Secondary Antibodies
    • Donkey anti-Rabbit IR800 (Licor)
    • Donkey anti-Goat IR680 (Licor)
    • Donkey anti-Mouse IR680 (Licor)

Notes for Teaching Faculty

TA notes, mod 2

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