Biochemistry Techniques Quiz

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Questions and Answers

What is the main purpose of using SDS in the electrophoresis process?

To selectively bind certain proteins

To facilitate the separation of nucleic acids

To denature proteins and add a negative charge (correct)

To increase the size of proteins

What is the fundamental difference between differential centrifugation and density gradient centrifugation?

Differential centrifugation is more accurate than density gradient centrifugation.

Differential centrifugation requires a longer spin time than density gradient centrifugation.

Differential centrifugation separates particles based on size, while density gradient separation is based on density. (correct)

Density gradient centrifugation uses a solid medium, while differential centrifugation does not.

What is the role of homogenization in protein analysis?

To stabilize proteins during the electrophoresis process

To purify proteins from other cellular components

To concentrate proteins for easier measurement

To break down cells and release their contents for analysis (correct)

In which stage of Western Blot analysis is the protein mixture separated by size?

Electrophoresis Signup and view all the answers

What type of gradients are typically used in equilibrium or density gradient centrifugation?

Pre-established sucrose gradients Signup and view all the answers

What is the primary purpose of the Western Blot technique?

To confirm the presence of a specific protein Signup and view all the answers

During a Western Blot, what process is referred to as 'blotting'?

Transferring proteins from a gel to filter paper Signup and view all the answers

What role do antibodies play in Western Blotting?

They help detect specific proteins in the sample Signup and view all the answers

How are proteins separated in a Western Blot analysis?

By molecular weight using SDS-PAGE Signup and view all the answers

Which step follows the transfer of proteins to filter paper in the Western Blot process?

Blocking the membrane to prevent nonspecific binding Signup and view all the answers

What does the term 'specificity' refer to in the context of Western Blotting?

The capability of an antibody to bind only to its target protein Signup and view all the answers

What is measured to determine the presence of a protein after a Western Blot?

The signal generated from the antibody interaction Signup and view all the answers

What does the thickness of the bands in a Western Blot indicate?

The relative amount of protein Signup and view all the answers

How is the membrane in a Western Blot generated?

By blotting proteins onto a membrane containing specific antigens Signup and view all the answers

What is the primary use of antibodies in a Western Blot?

To detect specific proteins or antibodies Signup and view all the answers

When conducting a Western Blot for HIV detection, what does the presence of HIV-antibodies indicate?

Previous exposure to HIV Signup and view all the answers

What is indicated by the density and darkness of bands in a Western Blot?

The relative amount of specific proteins Signup and view all the answers

What happens if the HIV-antibodies are absent in a patient's blood serum during a Western Blot?

No bands will be detected for HIV proteins Signup and view all the answers

In a Western Blot, what is the purpose of exposing the membrane to X-ray film?

To visualize the presence of proteins Signup and view all the answers

What type of proteins are typically used on the membrane in an HIV Western Blot?

HIV-specific proteins to capture antibodies Signup and view all the answers

Which step is fundamental to the detection of proteins in the Western Blot process?

Electrophoresis of protein samples Signup and view all the answers

Study Notes

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.

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Description

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.