Protein Fractionation by Centrifugation

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Questions and Answers

What does fractionation of proteins by centrifugation primarily separate?

Gaseous impurities in liquids

Liquid mixtures into solids

Heterogeneous particles in suspension (correct)

Homogeneous particles in solution

Which factor does NOT influence the rate of sedimentation in centrifugation?

Medium in which the particle is suspended

Temperature of the solution (correct)

Density of the particle

Mass of the particle

In what state are molecules best suited for centrifugation?

Concentrated state

Denatured state

Native state (correct)

Crystalline state

Which type of application is NOT typically associated with fractionation by centrifugation?

Purification of non-macromolecular fluids Signup and view all the answers

What purpose does analytical centrifugation serve?

To measure physical properties of small quantities Signup and view all the answers

What role do antibodies play in affinity chromatography?

They help purify specific proteins by binding to their antigens. Signup and view all the answers

How do antibodies contribute to the analysis of protein structure and function?

By providing a method for specific protein detection. Signup and view all the answers

What is the primary reason antibodies can bind to antigens with high affinity?

They have unique binding sites specific to each antigen. Signup and view all the answers

What is the approximate molecular weight of a light chain of an antibody?

~25kD Signup and view all the answers

What type of substance can antibodies target for purification in affinity chromatography?

Foreign substances known as antigens Signup and view all the answers

What enzyme is commonly used in ELISA for chromogenic detection?

Alkaline phosphatase Signup and view all the answers

What is the primary purpose of using specific antibodies in immunofluorescence microscopy?

To visualize changes in protein localization Signup and view all the answers

In the context of the immunolocalization of antigens, what may indicate protein relocation?

Movement from cytoplasm to nucleus Signup and view all the answers

Which method can be combined with fluorescent dyes for enhanced visualization in immunofluorescence?

Other fluorescent dyes Signup and view all the answers

What type of detection does ELISA primarily utilize?

Chromogenic, luminescent, or fluorescent detection Signup and view all the answers

What is a key characteristic of monoclonal antibodies?

They are derived from a single B-cell clone. Signup and view all the answers

What is the main advantage of using polyclonal antibodies as secondary antibodies in Western blotting?

They offer stronger signal amplification. Signup and view all the answers

What determines the specificity of polyclonal antibodies?

They recognize multiple different epitopes. Signup and view all the answers

What is involved in the Western blotting technique?

Separation of proteins based on size using SDS-PAGE. Signup and view all the answers

Which feature of immunoblotting enables the detection of protein-protein interactions?

Co-immunoprecipitation. Signup and view all the answers

How do polyclonal antibodies differ in their binding capabilities compared to monoclonal antibodies?

Polyclonal antibodies bind to multiple epitopes. Signup and view all the answers

Which of the following is NOT typically analyzed using Western blotting?

Lipids and carbohydrates in cells. Signup and view all the answers

What is commonly used as a membrane for transferring proteins in Western blotting?

Nitrocellulose or PVDF. Signup and view all the answers

What is the main goal of the protein purification workflow?

To isolate a specific protein or enzyme from a mixture Signup and view all the answers

Which method is commonly used in specific protein detection?

Immunological methods using specific antibodies Signup and view all the answers

What aspect of proteins can be monitored during purification?

Protein concentration or enzyme activity Signup and view all the answers

How can enzymatic activity be an indicator of specific proteins?

It reflects the ability of the enzyme to catalyze reactions Signup and view all the answers

In protein purification, what does fractionation refer to?

Separating the components of a mixture based on specific properties Signup and view all the answers

What is a key characteristic of antibodies used in protein detection?

They bind with high precision to specific proteins Signup and view all the answers

Which factor is essential for monitoring protein purification?

Gel electrophoresis technique Signup and view all the answers

What is one method of detecting proteins based on their interaction with ligands?

Affinity chromatography Signup and view all the answers

What is the main role of SDS in denaturing PAGE?

Denatures proteins by binding to hydrophobic residues Signup and view all the answers

How does SDS affect the charge of proteins?

It masks intrinsic charge, conferring a negative net charge Signup and view all the answers

What is the relationship between relative mobility and mass in denaturing PAGE?

Inverse relationship proportional to the logarithm of mass Signup and view all the answers

What type of bonds can be disrupted by SDS during PAGE?

Disulfide bonds and non-covalent interactions Signup and view all the answers

Which modification might cause deviations in mobility compared to standard mass estimates?

Extensive glycosylation Signup and view all the answers

A protein with many subunits would be most affected by which SDS action?

Dissociating multimeric proteins Signup and view all the answers

Why is SDS less effective for proteins with extensive membrane-spanning regions?

SDS only acts on water-soluble proteins Signup and view all the answers

Which factor is crucial for estimating the molecular mass of proteins after SDS-PAGE?

Comparison to standards of known mass Signup and view all the answers

Study Notes

Fractionation and Enrichment of Proteins by Centrifugation

Utilizes gravity to separate heterogeneous particles suspended in a solution.
Effective for isolating molecules in their native state, including protein complexes.
Sedimentation rate influenced by:
- Mass of the particle, impacting the force of gravity on its movement.
- Density of the particle, calculated as weight per volume.
- Properties of the medium, determining the resistance the particle encounters.

Applications of Centrifugation

Preparative Applications
- Facilitates the separation, fractionation, and resolution of large quantities of molecules.
- Useful in preparing samples for further analysis or experimentation.
Analytical Applications
- Allows measurement of physical properties of macromolecules, typically with smaller sample sizes.
- Serves as a tool for assessing characteristics such as sedimentation coefficients and molecular weights.

SDS-PAGE

SDS (sodium dodecyl sulfate) is an amphipathic detergent used in denaturing polyacrylamide gel electrophoresis (PAGE) to unfold proteins.
It disrupts non-covalent interactions such as hydrophobic, ionic, and van der Waals bonds, leading to the dissociation of multimeric proteins.
SDS binds to hydrophobic residues and reduces disulfide bonds, masking the intrinsic charge of proteins.
SDS binds at a ratio of ~1 SDS per 2 amino acids, making the charge-to-mass ratio uniform across different proteins.

Electrophoretic Mobility

Relative mobility of proteins in denaturing PAGE is inversely proportional to the logarithm of their mass, establishing a linear relationship for most proteins.
Deviations in mobility may occur due to modifications (e.g., glycosylation) or extensive membrane-spanning regions, aiding in molecular mass estimation against known standards.

Protein Purification Workflow

Workflow includes identifying the protein source, monitoring purification through gel electrophoresis, and analyzing protein structure and function.
Fractionation and enrichment steps improve protein yield and target protein concentration.

Specific Protein Detection

Proteins can be detected by their specific antibodies, enzyme activity, or ability to bind particular ligands.
Antibodies serve as highly selective molecules for identifying proteins, with various immunological methods available for detection.
Enzymatic activity can indicate the presence and function of specific proteins through observable reactions, often involving substrate color changes.

Antibodies (Immunoglobulins)

Antibodies are proteins produced by B-lymphocytes in response to antigens, exhibiting high specificity and affinity.
They play key roles in affinity chromatography for protein purification and analytical techniques.

Mono- and Polyclonal Antibodies

Monoclonal antibodies (MoAbs) are derived from a single B-cell clone, recognizing the same antigenic determinant.
Polyclonal antibodies (PAbs) are mixtures derived from multiple clones, recognizing different epitopes of the same antigen.

Western Blotting

Western blotting detects antigens after protein separation and transfer to a membrane, utilizing specific antibodies for visualization.
It can assess protein expression levels, modifications, degradation, and protein-protein interactions.

Immunoblotting Steps

Primary antibodies can be monoclonal or polyclonal; polyclonal antibodies are often preferred for their higher signal amplification and broader recognition.
Enzymes linked to secondary antibodies (like HRP or alkaline phosphatase) facilitate quantitation through chromogenic, luminescent, or fluorescent detection.

ELISA Formats

ELISA (Enzyme-Linked Immunosorbent Assay) enables detection and quantification of specific antigens or antibodies in samples, such as patient serum.

Immunofluorescence Microscopy

This technique detects and localizes proteins within cells or tissues using specific antibodies conjugated to fluorescent dyes.
It enables the observation of protein relocation between cellular compartments, visualized under fluorescence microscopy.

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Description

This quiz covers the principles and applications of protein fractionation and enrichment using centrifugation. It delves into how heterogeneous particles are separated by gravity based on their mass and density, including both preparative and analytical methods. Ideal for students interested in biochemistry and molecular biology.