44 Questions
Which of the following is true about immunofluorescence?
It uses antibodies to target fluorescent dyes to specific biomolecule targets.
What is the specific region on an antigen recognized by an antibody called?
Epitope
Which technique is immunofluorescence an example of?
Immunohistochemistry
Which type of immunofluorescence technique uses a single, primary antibody, chemically linked to a fluorophore?
Primary (direct)
What is the advantage of using secondary (indirect) immunofluorescence over primary (direct) immunofluorescence?
Signal amplification
Which microscope design is the simplest and widely used for analysis of immunofluorescence samples?
Epifluorescence
What is a disadvantage of direct immunofluorescence compared to indirect immunofluorescence?
Less sensitive
Which of the following is a limitation of immunofluorescence?
Autofluorescence
What is one way to control photobleaching in immunofluorescence?
All of the above
Why is immunofluorescence limited to fixed cells?
Antibodies cannot penetrate the cell membrane
What is the purpose of using recombinant proteins containing fluorescent protein domains in immunofluorescence?
To allow determination of protein localization in live cells
Which type of microscope has a higher resolution compared to light microscopes?
Transmission electron microscope (TEM)
Which development in electron optics allowed for the manipulation of the direction of an electron beam?
The work of Hertz in 1883
Which type of electron microscopy is used for chemical analysis?
Electron microprobe
Which team of researchers successfully generated magnified images of mesh grids placed over an anode aperture in 1931?
Adolf Matthias and Max Knoll
Who is considered the inventor of the electron microscope according to patent law?
Reinhold Rüdenberg
When did Ruska and Knoll build the first electron microscope that exceeded the resolution attainable with an optical microscope?
1933
Which company produced the first commercial electron microscope in 1938?
Siemens
Which of the following microscopes has the ability to determine the positions of atoms within materials?
Transmission electron microscope (TEM)
Which of the following is an advantage of electron diffraction over X-ray crystallography?
Electron diffraction does not require a single crystal or polycrystalline powder specimen
Which microscope design produces images with a single brightness value per pixel?
Scanning electron microscope (SEM)
Which microscope design has a greater depth of field and can produce images that represent the three-dimensional surface shape of a sample?
Scanning electron microscope (SEM)
Which of the following components is NOT typically found in a fluorescence microscope?
Objective lens
What is the purpose of the spectral emission filter in a fluorescence microscope?
To separate the illumination light from the emitted fluorescence
Which type of microscope design is commonly used for immunofluorescence samples?
Epifluorescence microscope
Which type of microscopy technique allows scientists to directly make a protein of interest fluorescent in biological samples?
Fluorescence microscopy
What is one limitation of fluorescence microscopy?
Fluorophores lose their ability to fluoresce as they are illuminated
Which microscopy technique allows observation of the specific structures that have been labeled for fluorescence?
Fluorescence microscopy
Which microscopy technique aims to reach past the diffraction limit by using specialized optical configurations?
Fluorescence microscopy
Which type of microscope design is commonly used for widefield epifluorescence microscopy?
Epifluorescence microscope
What is the main purpose of fluorescent staining in fluorescence microscopy?
To label specific proteins or molecules of interest
Which type of light source is commonly used for confocal microscopy and total internal reflection fluorescence microscopy?
Xenon arc lamps
What is the technique called that uses the specific binding of an antibody to label specific proteins or molecules within a cell?
Immunofluorescence
Which of the following is true about fusion proteins?
Fusion proteins are created through the joining of two or more genes that originally coded for separate proteins.
What is an example of an oncogenic fusion protein?
The bcr-abl fusion protein.
What can be the result of interactions between two proteins in a fusion protein?
Modification of their functions.
Which of the following is NOT a property that can be increased through fusion protein design?
Expression levels
What is the purpose of linker peptides in fusion proteins?
To improve protein purification
Which technique is commonly used for the identification and purification of proteins in fusion proteins?
Affinity chromatography
Which of the following fusion protein design techniques involves connecting the proteins of interest end-to-end?
Tandem fusion
Which of the following is true about chimeric protein drugs?
Chimeric protein drugs are created through the fusion of two different proteins.
What is the advantage of using photoconvertible fluorescent proteins (PCFPs) as tags in protein tracking?
PCFPs allow for real-time tracking of protein interactions.
What is the purpose of creating recombinant fusion proteins?
To combine properties from two different proteins into a single chimeric protein.
How are humanized antibodies different from chimeric antibodies?
Humanized antibodies have replaced segments of the antibody molecule with human portions.
Study Notes
Immunofluorescence
- Immunofluorescence is a technique that uses antibodies or immunoglobulins to detect specific proteins in cells.
- The specific region on an antigen recognized by an antibody is called an epitope.
Types of Immunofluorescence
- Direct immunofluorescence uses a single, primary antibody chemically linked to a fluorophore.
- Indirect immunofluorescence uses a primary antibody and a secondary antibody conjugated to a fluorophore.
- The advantage of using secondary (indirect) immunofluorescence over primary (direct) immunofluorescence is that it increases sensitivity and flexibility.
Microscope Designs
- The simplest and widely used microscope design for analysis of immunofluorescence samples is the epifluorescence microscope.
- Confocal microscopy has a higher resolution compared to light microscopes.
- Electron microscopy has a higher resolution compared to light microscopes.
- Scanning electron microscopy (SEM) has a greater depth of field and can produce images that represent the three-dimensional surface shape of a sample.
Limitations and Controls
- A disadvantage of direct immunofluorescence compared to indirect immunofluorescence is lower sensitivity.
- A limitation of immunofluorescence is photobleaching, which can be controlled by reducing light intensity, using antioxidants, or taking images quickly.
- Immunofluorescence is limited to fixed cells because the procedure requires cell fixation to preserve antigenicity.
Recombinant Proteins and Fusion Proteins
- Recombinant proteins containing fluorescent protein domains are used in immunofluorescence to create fusion proteins.
- The purpose of using recombinant proteins is to create fluorescently labeled proteins for tracking and detection.
- An example of an oncogenic fusion protein is BCR-ABL1, which is a hallmark of chronic myeloid leukemia.
- Fusion proteins can lead to changes in protein function, localization, or stability.
- Linker peptides are used in fusion protein design to connect proteins of interest.
Microscopy Techniques
- Confocal microscopy uses a laser and pinhole to produce images with a single brightness value per pixel.
- Total internal reflection fluorescence microscopy (TIRFM) uses an evanescent wave to produce images near the surface of a sample.
- Fluorescence microscopy is limited by the diffraction limit, which can be overcome by using specialized optical configurations (super-resolution microscopy).
- Chimeric proteins are generated by combining parts of two proteins from different species.
Antibody Drugs and Proteins
- Chimeric protein drugs are used to reduce immunogenicity and increase efficacy.
- Humanized antibodies are different from chimeric antibodies in that they have more human-derived components.
- Photoconvertible fluorescent proteins (PCFPs) are used as tags in protein tracking due to their ability to change fluorescence emission wavelengths.
- The purpose of creating recombinant fusion proteins is to study protein function, localization, and interactions.
Test your knowledge on Immunofluorescence and its applications in biological sample analysis. Learn about the specificity of antibodies, targeting fluorescent dyes, and visualizing target molecules within cells.
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