Congratulations on finishing your final experiment in 20.109! The goal for today is to analyze the CETSA results and get a strong start on your Mini-report assignment. Please use your time wisely today.
Part 1: Analyze CETSA data
Import image file
The first thing you will need to do is import your entire Licor image file into the Image Studio workspace.
- Download the zip file of your data onto your computer
- Open Image Studio and select your workspace
- Click on the yellow circular IS button and select Import -> Import Studio Zip File
- Select your zip file
- 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
- 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.
- Select the Shapes tab on the bottom menu below the image
- Center your bands of interest in the image window and make them large enough to see gaps in between bands
- Open the Analysis menu and select Draw Rectangle
- 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. Why would that be?)
- Once rectangle is drawn, measurements will appear in Shapes tabs and the rectangle on the image will have a corresponding number and signal value
- 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.
- Click in center of the next band to add a rectangle of the same area to that band (Why should the rectangles have the same area?)
- If the rectangle doesn’t encompass the entire band, delete it and try again
- Continue this process until all bands have rectangles and data points in the Shapes Tab
- Copy data from Shape tab to Excel and label each row with corresponding treatment condition
- Select the 800 channel and repeat the process
Identify band quality
- With band of interest selected, chose "Profiles" on the vertical tab on the right-hand side of the screen (this menu tab should currently have "Display" selected). You will see a peak that shows the image signal.
- 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. What do each of these peak components tell you about the signal quality in the Western blot?
Part 2: Begin Mini-report assignment
The Mini-report is the final assignment you will complete for 20.109! The format is a short write-up of the data you collected in Mod 3 and the logistics can be found on the Mini-report assignment page.
This assignment should not require a lot of your time. Ideally, you will be able to finish the bulk of the data analysis and some of the writing in class today. Below is a list of the things that should be included:
- The chemical structure of your ligand.
- In the text you should included some information on why you propose this ligand is a possible binder of FKBP12. This can be related to the structural features or based on the data collected in previous semesters.
- The Western blot image.
- This is a data figure and should be presented like the data figures in your previous assignments.
- Analysis of the Western blot image.
- Please compare treatment conditions to the unheated DMSO lane and report the ratio.
- The details of this can be included as text or as a figure.
- Table comparing Tm data for all of the ligands tested by the class.
- In the text you should make a conclusion regarding which ligands are / are not binders and why.
Next day: Research proposal presentations
Previous day: Complete antibody staining for Western blot analysis