Difference between revisions of "20.109(S23):M2D7"

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(Introduction)
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*What is a benefit of using a lower objective for this imaging?  A consequence?
 
*What is a benefit of using a lower objective for this imaging?  A consequence?
 
*Where do you expect to see the 568 nm signal in your images?  What might it mean if you don't see the signal where it is expected?  What might it mean if you see the signal where it is not expected?
 
*Where do you expect to see the 568 nm signal in your images?  What might it mean if you don't see the signal where it is expected?  What might it mean if you see the signal where it is not expected?
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===Part 2: Assess Fet4 expression===
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===Part 3: Perform cytotoxicity experiment===
  
 
==Navigation links==
 
==Navigation links==
 
Next day: [[20.109(S23):M2D8 |IF staining and prepare for cytotoxicity experiment]] <br>
 
Next day: [[20.109(S23):M2D8 |IF staining and prepare for cytotoxicity experiment]] <br>
 
Previous day: [[20.109(S23):M2D6 |Complete data analysis and organize Research article figures]] <br>
 
Previous day: [[20.109(S23):M2D6 |Complete data analysis and organize Research article figures]] <br>

Revision as of 17:16, 3 February 2023

20.109(S23): Laboratory Fundamentals of Biological Engineering

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Spring 2023 schedule        FYI        Assignments        Homework        Class data        Communication        Accessibility

       M1: Drug discovery        M2: Protein engineering        M3: Project design       

Introduction

Protocols

Part 1: Image Fet_mutant expression experiment

Diagram of a fluorescence microscope.
As discussed in prelab, two antibodies were used in the H2AX assay. The first antibody, or primary antibody, was anti-His tag and raised in a mouse. The secondary antibody was anti-mouse and raised in a goat, more importantly, this molecule is conjugated to a fluorescent dye tag called Alexa Fluor 568. The Alexa Fluor 568 tag is a orange/red fluorescent dye that is excited at 568 nm. To visualize the expression of your tagged mutant Fet4, we will use fluorescence microscopy.

In fluorescence microscopy the specimen is illuminated with a wavelength of light specific to the excitation of the fluorescent tag used to target the feature of interest. The excitation wavelength is absorbed by the fluorescent tag, which causes it to emit light at a longer, less energetic wavelength. Typically, fluorescence microscopes used in biology are an epifluorescence type with a single light path (the objective) for excitation and emission detection, as depicted in the diagram above.

Fluorescence, or epifluorescence, microscopes are composed of a light source, an excitation filter, a dichroic mirror, and an emission filter. The filters and the dichroic mirror are specific to the spectral excitation and emission characteristics of the fluorescent tag. To visualize fluorescence, light at the excitation wavelength is focused on the sample. The light emission from the sample is focused by the objective to a detector.

Due to timing reasons, the Instructors will complete the imaging for the coverslips prepared during this laboratory session. To ensure you are familiar with the imaging procedure, each team will see a demonstration provided by the Instructor.

In your laboratory notebook, complete the following:

  • What is the type of microscope used?
  • What is the light source used by this microscope?
  • Which objective are you using to image the yeast?
  • What is a benefit of using a higher objective for this imaging? A consequence?
  • What is a benefit of using a lower objective for this imaging? A consequence?
  • Where do you expect to see the 568 nm signal in your images? What might it mean if you don't see the signal where it is expected? What might it mean if you see the signal where it is not expected?


Part 2: Assess Fet4 expression

Part 3: Perform cytotoxicity experiment

Navigation links

Next day: IF staining and prepare for cytotoxicity experiment

Previous day: Complete data analysis and organize Research article figures