MCB 253 Final Exam Study Guide

Techniques Materials Examples of what is expected

Protein ❑ Principles behind the technique ❑ What is the purpose of a Western blot?
Characterization: ❑ General protocols ❑ What steps does a Western blot analysis entail?
Western Blot with ❑ Expected outcomes ❑ What are the steps for creating the blotting sandwich?
detection ❑ Various reagents and their purpose ❑ What causes proteins to migrate from the gel to the
❑ MW standard nitrocellulose during Western blot transfer? Which
direction do proteins move?
❑ Properties of the protein that each
❑ Why do you have to use a protein MW marker (ladder) when
technique relies on
performing a Western blot?
❑ Different types of controls that can be used
❑ What are the differences between imaging a protein on a gel
to analyze WB results
vs. a nitrocellulose membrane?
❑ Expression levels of proteins in cell lines
❑ What do you expect to see on a WB nitrocellulose membrane
❑ Results from different types of samples on
after it is developed?
a WB
❑ What is the difference between a primary and a secondary
❑ Specificity of primary antibodies, specificity
antibody? Monoclonal vs. polyclonal Abs?
of secondary antibodies their detection
❑ What is the HRP color development reaction? How does it
principle
work?
❑ Why would someone use Ponceau S instead of Ab/HRP color
development, or coomassie?
❑ How would a specific positive/negative control help analyze data?
❑ Why can one antibody show multiple bands?
Cellular ❑ Fluorescence basics- fluorophores, ❑ What is fluorescence? What is a fluorophore? What is
Localization: excitation/ emission filters/ wavelengths immunofluorescence?
Fluorescence ❑ Different types of probes and what they ❑ What are the similarities/differences between
detect Rhodamine Phalloidin, WGA, and DAPI?
❑ Cell lines/ types, confluency, cell count ❑ What does WGA bind to? What does DAPI bind to?
❑ Visualization/microscopy ❑ How does fluorescent microscopy work?
❑ Imaging- viewing area, ❑ What are the functions of a ZOE Cell Imager?
scale/magnification, merge images ❑ What information can be obtained from a given fluorescence
image/merge image?

All of the following could be used to develop exam questions:

1) All required readings from the Lodish textbook and the MCB 253 Lab Manual
2) All readings/documents posted in the MCB 253 Moodle (you do NOT need to know the information in the COVID-19 PPTs)
3) All protocols/experimental techniques used in the course (including purpose, reagents and their functions, lab equipment, data analysis, etc.)
Additional Information:
There will be 30 multiple-choice questions (25 questions x 3 points each, 75 points total AND 5 questions x 5 points each, 25 points total); the exam is
worth 100 points total. The exam will be administered online in LON CAPA. Your TA will proctor you using Zoom.