Biochemistry Techniques Quiz

Topic I – Studying Cells - Techniques

• A handout describes how to take notes from lectures. Lecture outlines are available online, but students must attend to get complete notes.
• Students should bring posted figures and outlines to class.

Objectives

• Classify different types of cells
• Outline the basic principles of microscopy
• Explain different types of microscopy
• Describe the advantages and disadvantages of using stains to study cells
• Describe recent advances in studying and visualizing live cells
• Explain the purpose of differential centrifugation and sedimentation
• Outline steps in a Western blot
• Determine the best technique for a given cellular question

1. What are Cells?

• The Cell Theory, proposed by Schleiden and Schwann in 1839, states all organisms consist of cells, and the cell is the basic unit of life.
• All cells arise from preexisting cells.
• Two broad groups of cells–Prokaryotic (Bacteria and Archaea) and Eukaryotic.
• Prokaryotic cells do not have membrane-bound organelles, while Eukaryotic cells contain membrane-bound organelles and a true nucleus

Cell Size

• Micrometers(µm) and nanometers(nm) are used to measure cell sizes.
• Typical plant and animal cells are 20-30 µm; Bacteria are 1-2 µm
• Ribosomes are 25 nm; Microfilaments are 7 nm

Microscopy

• Cell structures are often too small to be seen with the naked eye.

• Different types of microscopes are used for different tasks, and there is always a tradeoff between resolution and what can be seen. Different microscopes allow analysis of different levels of structure in the cell, and the type of microscope will influence what information can be seen.

• Light microscopy

  • Resolution: the minimum distance between two objects to be distinguished as separate.
  • Human eye resolution = ~ 90 µm (0.09 mm)
  • Light microscope resolution = ~200 nm (0.2 µm)
  • Electron microscope resolution= ~0.2 nm

• Interference microscopy (DIC): Increases image contrast to be able to better visualize the cell's finer structures

• Fluorescence microscopy uses fluorescent molecules to highlight specific structures and proteins within cells, allowing detailed analysis of cellular processes.

• Immunofluorescence uses antibodies tagged with fluorescent molecules to pinpoint specific proteins or antigens within a cell.

Additional Techniques

• Confocal microscopy uses a laser to illuminate a thin plane of a specimen, enabling visualization of structures within thick specimens.
• Electron microscopy has much higher resolution (~0.2nm) than light microscopy but is limited to observing dead specimens.
• Scanning EM: scans the surface of a specimen to get a 3D image
• Transmission EM: sends electrons through a specimen to see its internal structures.
• Cell homogenization prepares tissues or cells to separate contents (centrifugation, enzymatic treatment, sonication etc)

Differential Centrifugation

• Separates cell components according to density.
• This technique separates cellular components by generating a gradient of density, utilizing the differing sedimentation rates that arise due to density differences.

Studying Proteins

• Proteins are complex and essential for cellular function.
• Electrophoresis separates proteins based on size.
• SDS-PAGE is a specific technique for separating proteins by size. SDS is a detergent that unfolds proteins and adds a net negative charge.
• Western blotting separates proteins based on size via electrophoresis first and then labels specific proteins with antibodies.

Test your knowledge on key biochemistry techniques such as SDS electrophoresis, centrifugation methods, protein analysis, and Western Blotting. This quiz covers fundamental concepts and differences in methodology used in molecular biology labs. Perfect for students and professionals in biochemistry and related fields.