Difference between revisions of "20.109(S21):M2D7"

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===Part 2: Analyze CETSA data===
===Part 2: Analyze CETSA data===
Download Image Studio Lite, the software used the scan your blots on Licor [https://www.licor.com/bio/image-studio-lite/download link here]:
''Import image file into the Image Studio workspace.''
''Import image file into the Image Studio workspace.''

Revision as of 14:07, 22 April 2021

20.109(S21): Laboratory Fundamentals of Biological Engineering

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Spring 2021 schedule        FYI        Assignments        Homework        Communication |        Accessibility

       M1: Antibody engineering        M2: Drug discovery        M3: Protein engineering       


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-TDP43 and α-tubulin antibodies and then scan the Western blots using a specially constructed microscope located in the Lauffenburger lab.

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.


Part 1: Probe blot to finish CETSA experiment

To ensure the steps included below are clear, please watch the video tutorial linked here: [Western Blot]. The steps are detailed below so you can follow along!

  1. The blot that you prepared in the previous laboratory session was stored in the 4 °C cooler in blocking buffer.
  2. Retrieve your blot from the front laboratory bench.
  3. Prepare the primary antibody solution.
    • Dilute the primary antibodies in 10 mL of blocking buffer.
    • For the α-TDP43 you will use a rabbit anti-α-TDP43 antibody at a 1:1000 dilution.
    • For the α-tubulin you will use mouse anti-α-tubulin antibody at a 1:5,000 dilution.
  4. Pour off the blocking solution in the sink.
  5. Add the primary antibody solution to your blot.
  6. Carefully place the blot on the rotating table and cover with aluminum foil.
  7. Shake at 65 rpm for 60 min.
  8. Obtain your blot from the rotatating table and pour the primary antibody solution into a conical tube.
    • Label with team and section information.
    • Because the antibody is in excess, the diluted primary solution can be re-used and is worth saving until you see the results of your Western blot.
  9. Add enough TBS-T to cover the membrane.
  10. Return your blot to the rotating table for 5 min at 80 rpm.
  11. Repeat for a total of 3 washes.
  12. Immediately before pouring off the last wash, prepare the secondary antibody solution.
    • Dilute the secondary antibodies in 10 mL of blocking buffer.
    • For the α-TDP43 (raised in a rabbit) you will use the donkey anti-rabbit IR680 (RED) antibody at a 1:10,000 dilution.
    • For the α-tubulin (raised in a mouse) you will use the goat anti-mouse IR800 (GREEN) antibody at a 1:10,000 dilution.
    • The secondary antibodies are light sensitive and should be kept in the dark! Use aluminum foil to wrap your tube.
  13. Add the secondary antibody solution to your blot.
  14. Carefully place the blot on the rotating table and cover with aluminum foil.
  15. Shake at 65 rpm for 60 min.
  16. Pour off the secondary antibody in the sink.
  17. Wash the membrane by adding TBS-T and shake for 5 min at 80 rpm, using the rotating table.
  18. Repeat for a total of 3 washes.
  19. The Odyssey scanner is located in the Lauffenburger laboratory, one of the teaching faculty will accompany you there to scan your blot.

To ensure you are familiar with the steps involved in imaging the CETSA experiment, please watch the video tutorial linked here: [Western Blot Imaging].

Part 2: Analyze CETSA data

Download Image Studio Lite, the software used the scan your blots on Licor link here:

Import image file into the Image Studio workspace.

  1. Download the zip file for the Western blot of one of the small molecules you selected in the previous laboratory session onto your computer.
    • Access the Western blot data here.
  2. Open Image Studio and select your workspace.
  3. Click on the yellow circular IS button and select Import -> Import Studio Zip File.
  4. Select your zip file.
  5. The image of your blot should appear with both the red 700 channel and the green 800 channel. If you are unable to see all of your bands, zoom out to 75%.

Analyze single channel band intensity.

  1. Select the 700 Channel on the display menu. The 800 channel should not be present in the image and the 800 segment should be a gray box with a line through it in the display menu.
  2. Select the Shapes tab on the bottom menu below the image.
  3. Center your bands of interest in the image window and make them large enough to see gaps in between bands.
  4. Open the Analysis menu and select Draw Rectangle.
  5. Draw a rectangle around the first band.
    • The rectangle should be big enough to fully encompass each band in the row, but do not allow the rectangle to overlap multiple bands
    • Note: the numbers on the shapes tab will change depending on rectangle size.
  6. Once the rectangle is drawn, measurements will appear in Shapes tab and the rectangle on the image will have a corresponding number and signal value.
  7. To measure next band, make sure the rectangle is a hashed line rather than a solid line, then go to the Analysis menu and select Add Selection.
    • If you accidentally de-select the rectangle (line goes from dotted to solid), the software won’t allow you to Add Selection. Do not draw a new rectangle. Instead change cursor to the “select” cursor and select the currently drawn box to take the line back to dashed. Then click Add Selection and continue.
  8. Click in center of the next band to add a rectangle of the same area to that band.
    • If the rectangle doesn’t encompass the entire band, delete it and try again.
  9. Continue this process until all bands have rectangles and data points in the Shapes tab.
  10. Copy data from Shape tab to Excel and label each row with corresponding treatment condition.
  11. Select the 800 channel and repeat the process.

In your laboratory notebook, complete the following:

  • When drawing the rectangle in Step #5, the numbers it was noted that the numbers change in the Shapes tab depending on the rectangle size. Why is this the case?
  • Why is it important that all of the rectangles used to measure the bands on the Western blot have the same area?

Examine band quality

  1. With the band of interest selected, chose "Profiles" on the vertical tab on the right-hand side of the screen.
    • This menu tab should have "Display" selected.
    • You will see a peak that shows the image signal.
  2. High quality fluorescent band peaks share some characteristics with high quality Sanger sequencing peaks. The peak should be clear and the base of the peak should intersect with the baseline present on the peak graph.

In your laboratory notebook, complete the following:

  • What do each of the peak components tell you about the signal quality of the band in the Western blot?

Part 3: Complete CETSA data analysis

Plot your CETSA data such that the intensity of the bands from the CETSA experiment are shown on the y-axis for each of the conditions tested, which should be shown on the x-axis. Graph the results for each of the small molecules separately.

In your laboratory notebook, complete the following:

  • Include the plots for your CETSA data.
  • Describe the results for the CETSA experiments. What do the data show? What do the data indicate?
  • Are the results unexpected or unclear? If so, how might you improve the experiment or change the conditions used to clarify the results? If not, what is a potential next step in testing the small molecule?

Reagents list

  • Odyssey blocking buffer (from Licor)
  • α-TDP43 primary antibody (from ProteinTech)
  • α-tubulin primary antibody (from Cell Signaling Technologies)
  • donkey anti-rabbit IR680 secondary antibody (from Licor)
  • goat anti-mouse IR800 secondary antibody (from Licor)

Navigation links

Next day: Review inverse pericam (IPC) cloning strategy

Previous day: Utilize cellular thermal shift assay (CETSA) to test putative small molecule binders