20.109(S17):Complete Western blot and induce DNA damage for survival and quantitative PCR assays (Day2)

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20.109(S17): Laboratory Fundamentals of Biological Engineering

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Schedule Spring 2017        Announcements        Assignments        Homework        Communication
       1. High-throughput ligand screening        2. Gene expression engineering        3. Biomaterials engineering              

Introduction

In the previous laboratory session you isolated the protein fraction from cell lysates. Today you will separate the proteins in the lysates using a polyacrylamide gel and transfer the proteins onto a nitrocellulose membrane. This step enables you to 'probe' the protein fractions isolated from cells for your protein of interest, which in our experiment is BRCA2. To probe the membrane, it was incubated with two primary antibodies overnight by the teaching faculty - α-BRCA2 and α-α-tubulin. The α-BRCA2 antibody was raised in a XXXXX and the α-α-tubulin antibody was raised in a rabbit. Primary antibodies bind directly to the protein of interest. Today you will add a secondary antibodies. Each secondary antibody will bind to the appropriate primary antibody and provide a means for visualization - in our experiment, the secondary antibody is responsible for a fluorescent signal...think back to the anti-His antibody used in module 1!

The ability to bind specific proteins using antibodies, or immunoglobulins, is critical in Western blot analysis. Antibodies are typically 'raised' in mammalian hosts. Most commonly mice, rabbits, and goats are used, but antibodies can also be raised in sheep, chickens, rats, and even humans. 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. 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. Antibodies can be raised not only to detect specific amino acid sequences, but also post-translational modifications and/or secondary structure. Therefore, antibodies can be used to distinguish between modified (for example, phosphorylated or glycoslyated proteins) and unmodified protein.

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. In this process, normal antibody-producing B cells are fused with immortalized B cells, derived from myelomas, by chemical treatment with a limited efficiency. To select only heterogeneously fused cells, the cultures are maintained in medium in which myeloma cells alone cannot survive (often HAT medium). Normal B cells will naturally die over time with no intervention, so ultimately only the fused cells, called hybridomas, remain. A fused cell with two nuclei can be resolved into a stable cell line after mitosis.

Strategy for generating monoclonal 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 the purity (that it has few other antibodies), the specificity (that it recognizes the antigen and not other spurious proteins) and the concentration (sometimes called 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. The purity, specificity and titer of the new antiserum will likely differ from those 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.

Strategy for generating polyclonal antibodies.


For Western blot 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 polyacrylamide gels, the protein capacity for each lane is 100 to 200 μg (that would be 20 μL of a 5-10 μg/μL protein preparation). Thus, 1 ng represents a protein that is approximately 0.0005-0.001% of the total cellular protein (1 ng out of 100,000-200,000 ng). Proteins that make up a more significant fraction of the total protein population will be easier to detect.

Traditionally, only one species of antibody could be used on a Western blot because the detection relied on the emission of light that was collected by x-ray film. In the traditional systems the output looks like black bands on a blue or clear background. However, more recent conjugate chemistry has allowed secondary antibodies to be coupled to fluorescent tags. Today we will use infrared (IR) secondary antibodies to detect our α-BRCA2 and α-tubulin antibodies and then scan the Western blots using a specially constructed microscope located in the Lauffenburger lab to determine the level of BRCA2 in our cell lines.

Licor detection of IR-dye conjugated antibodies. Image was modified from the Odyssey manual.
The Licor Odyssey scanner consists of an inverted microscope with two lasers that excite dyes which emit light in the IR range. As depicted in the image on the right, an excitation point is created when beams from the 700 nm and 800 nm lasers (A) are focused on the scanning surface. The microscope objective (B) is focused on the excitation point and collects light from the fluorescing IR dyes. This light is passed through a dichroic mirror (C) that separates the light into two distinct signals that travel through two independent optical paths that are focused on separate silicon photodiodes (D) and detected. In the image, the first channel (E) and second channel (F) are shown separately and merged (G).

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.

Protocols

Part 1: Induce DNA damage

Researchers often work on multiple experiments at the same time. For this to be fruitful, it is critical that they have a plan of action at the start. Today you will complete steps for both the survival assay and the quantitative PCR assay in tissue culture. Carefully read through Part 1a and Part 1b below to make a strategy with your partner...perhaps each team member can complete one exercise.

For both exercises, you will first induce DNA damage by incubating your cells with etoposide. Etoposide is itself a chemotherapy drug that generates DNA stand breaks by forming a ternary complex with DNA and topoisomerase II. This prevents religation of the DNA strands after unwinding. This effects cancer cells more than non-cancerous cells because cancer cells divide much faster. We will use etoposide to damage the DNA and then treat with an additional NHEJ-targeting drug, either mibefradil or loperamide. The mechanism by which mibefradil and loperamide target NHEJ is unknown (and therefore one of our research questions), though both drugs are useful in the treatment of other conditions:

  • Mibefradil is is a calcium channel blocker used to treat hypertension and chronic angina pectoris.
  • Loperamide is known to slow the contractions of the intestines and is used to treat gastroenteritis, inflammatory bowel disease, and short bowel syndrome.

Before you begin, select which drug (mibefradil or loperamide) you will use for your experiments in this module and enter your team information in the table below.


T/R section

Mibefradil Loperamide
1
2
3
4


W/F section

Mibefradil Loperamide
1
2
3
4

Part 1a: Treat cells for survival assay

To measure cell survival, you will complete a crystal violet staining assay. Crystal violet is a dye that binds negatively charged molecules. When a solution of crystal violet is mixed with cultured cells, the dye will bind to the proteins and carbohydrates in the membrane. Therefore, the amount of dye adsorbed can provide information concerning the number of cells that are present.

Sp17 20.109 M2D1 cell survival.png

In this exercise, you will induce DNA damage in the cells that you seeded during the previous laboratory session then add the drug that you selected above. Next class you will examine survival of your cells in response to the drug treatment.

  1. Prepare your working space within the tissue culture hood.
  2. Calculate the volume of etoposide stock needed for DNA damage induction.
    • Obtain an aliquot of pre-warmed media from the 37 °C water bath (11 mL).
    • Determine the volume of etoposide stock (100 mM) you need to add to the media for a final concentration of 100 μM.
  3. Retrieve your 12-well plates from the 37 °C incubator and visually inspect your cells with a microscope.
    • Record your observations concerning media color, confluency, etc. in your laboratory notebook.
  4. Move your plate into the tissue culture hood.
  5. Aspirate the spent media from each well.
    • Be careful not to cross-contaminant between wells with different cell lines!
  6. Add ~1 mL of PBS to each well and rock the plate gently to wash the cells.
  7. Aspirate the PBS from each well.
    • Again, be careful not to cross-contaminate.
  8. Add 1 mL of PBS to well A1 and A3.
    • These wells will be the 'no DNA damage' controls for the DLD-1 and BRCA2- cells.
  9. Add 1 mL of the media containing etoposide that you prepared in Step #2 to the empty wells.
  10. Carefully put your plate in the 37 °C incubator for 60 min.
  11. Assist you partner if they are still working. When you are both done, return to the main laboratory space.
  12. Return to the tissue culture space and retrieve your plate from the 37 °C incubator.
  13. Aspirate the PBS or media containing etoposide from each well.
    • Be mindful of cross-contamination.
  14. Add 2 mL of fresh media to each well.
  15. Calculate the volume of drug you need to add to each well for the final concentrations shown on the figure below.
    • Be sure to use the concentrations appropriate for the drug you chose.
    • The stock concentrations of mibfradil and loperamide are 10 mM.
    • It may be helpful to dilute the drug stocks to avoid pipetting very small volumes.
  16. Add the appropriate volume of drug and move your 12-well plate to the 37 °C incubator.
  17. Assist your laboratory partner, if necessary, then clean the hood and return the main laboratory space.
Sp17 20109 M2D2 drug plate map.png

Part 1b: Treat cells for quantitative PCR assay

To measure gene expression of DNA cell cycle checkpoint factors, you will perform quantitative PCR (qPCR). The presence of checkpoint factors in an indication of DNA damage in that the cell cycle is halted when excessive damage is incurred by a cell. In qPCR, the amount of a specific transcript can be measured. We will discuss the details more on M2D9.

Sp17 20.109 M2D2 qPCR.png

In this exercise, you will induce DNA damage in cells that were seeded by the teaching faculty then add the drug that you selected above. Next class you will purify RNA from your cells for quantitative PCR analysis.

  1. Prepare your working space within the tissue culture hood.
  2. Calculate the volume of etoposide stock needed for DNA damage induction.
    • Obtain an aliquot of pre-warmed media from the 37 °C water bath (9 mL).
    • Determine the volume of etoposide stock (100 mM) you need to add to the media for a final concentration of 100 μM.
  3. Retrieve four T25 flasks (two DLD-1 cultures and two BRCA2- cultures) from the 37 °C incubator and visually inspect your cells with a microscope.
    • Record your observations concerning media color, confluency, etc. in your laboratory notebook.
  4. Move your flasks into the tissue culture hood.
  5. Aspirate the spent media from each flask.
    • Be careful not to cross-contaminant between flasks with different cell lines!
  6. Add ~2 mL of PBS to each flask and rock the plate gently to wash the cells.
  7. Aspirate the PBS from each flask.
    • Again, be careful not to cross-contaminate.
  8. Add 2 mL of the media containing etoposide that you prepared in Step #2.
  9. Carefully put your flasks in the 37 °C incubator for 60 min.
  10. Assist you partner if they are still working. When you are both done, return to the main laboratory space.
  11. Return to the tissue culture space and retrieve your flasks from the 37 °C incubator.
  12. Aspirate the media containing etoposide from each flask.
    • Be mindful of cross-contamination.
  13. Add 3.5 mL of fresh media to each flask.
  14. Label one DLD-1 and one BRCA2- flask '+etop' to denote that the cells were treated with etoposide. Move these flasks to the 37 °C incubator.
    • These cultures are the 'no drug treatment' controls.
  15. Label the remaining two flasks '+etop, +mib' or '+etop, +lop' depending on which drug you selected.
  16. Calculate the volume of drug you need to add to your flasks for a final mibefradil concentration of 4.1 μM or loperamide concentration of 2.8 μM.
    • Be sure to use the concentrations appropriate for the drug you chose.
    • The stock concentrations of mibfradil and loperamide are 10 mM.
    • It may be helpful to dilute the drug stocks to avoid pipetting very small volumes.
  17. Add the appropriate volume of drug and move your flasks to the 37 °C incubator.
  18. Assist your laboratory partner, if necessary, then clean the hood and return the main laboratory space.

Part 2: Complete Western blot

Last time, you prepared protein extracts from DLD-1 and BRCA2- cells, separated them using SDS-PAGE, and then transferred them to a nitrocellulose membrane. Following the transfer step, blots were moved to blocking buffer. The next day, the teaching faculty added the α-BRCA2 and α-tubulin primary antibodies and incubated the membranes overnight at 4 °C.

  1. Obtain your blots from the front bench. Pour the antibody solution into a conical tube, writing the identity of the antibodies and the date on the tube.
    • Because the antibody is in excess, the diluted primary solution may be re-used on another blot and is thus worth saving until you see your Western blot.
  2. Add enough TBST to cover your membrane - no need to measure a volume.
    • 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!
    • TBST is Tris-buffered saline with 0.1% Tween 20 (a surfactant).
  3. Shake your container for 5 min at 80 rpm using the room temperature shaker.
  4. Repeat for a total of 4 washes.
  5. Just before pouring off the last wash, prepare the secondary antibodies.
  6. Dilute the secondary antibodies in 10 mL of Blocking Buffer.
    • They are light sensitive so find them on the front bench next to the Blocking Buffer and then wrap your tube in aluminum foil.

#For DNA-PKcs (mouse)/ α-tubulin (rabbit) -- use the goat anti-mouse IR800 (GREEN) antibody at 1:10,000 + donkey anti-rabbit IR680 (RED) at 1:10,000.

  1. After the last wash, add your secondary antibody solution, place on the room temperature shaker, and cover your Western blot container with aluminum foil.
  2. Shake at 65 rpm for 60 min.
  3. Pour off the secondary antibody in the sink.
  4. Wash the membrane by adding TBST and shake for 5 min at 80 rpm, using the room temperature shaker.
  5. Repeat for a total of 4 washes.
  6. The Odyssey scanner is located in the Lauffenburger laboratory (56-378), one of the teaching faculty will accompany you there in groups to scan your blots.

Part 3: Discuss research paper

We will end today with a discussion of the Dietlein et al. research article. In their research, the authors completed a screen to examine 1319 cancer-associated genes from 67 cell lines to identify cancer-cell specific mutations that are associated with DNA-PKcs dependence or addiction. Refer to the M2 overview for more information on addiction in cancer cells.

Our paper discussion will assist you in writing a cohesive story that clearly reports the data and provides strong support for the conclusions made about the data. During the paper discussion, everyone is expected to participate - either by volunteering or by being called upon!

Introduction

Remember the key components of an introduction:

  • What is the big picture?
  • Is the importance of this research clear?
  • Are you provided with the information you need to understand the research?
  • Do the authors include a preview of the key results?

Results

Carefully examine the figures. First, read the captions and use the information to 'interpret' the data presented within the image. Second, read the text within the results section that describes the figure.

  • Do you agree with the conclusion(s) reached by the authors?
  • What controls are included and are they appropriate for the experiment performed?
  • Are you convinced that the data are accurate and/or representative?

Discussion

Consider the following components of a discussion:

  • Are the results summarized?
  • Did the authors 'tie' the data together into a cohesive and well-interpreted story?
  • Do the authors overreach when interpreting the data?
  • Are the data linked back to the big picture from the introduction?

Reagents

Navigation links

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