Biochemistry 14.3 : Chromatography Basics
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Questions and Answers

What is the purpose of chromatography in biochemical analysis?

  • To purify and separate proteins or other biomolecules (correct)
  • To express proteins in large amounts
  • To visualize the sequence of amino acids in proteins
  • To analyze the genetic content of cells

Which of the following best describes the stationary phase in chromatography?

  • The endpoint of the chromatography process
  • The molecules of interest themselves
  • Small beads or gel packed within a column (correct)
  • The solution that carries molecules of interest

How do molecules interact with the stationary phase in chromatography?

  • All molecules move through the stationary phase at the same speed
  • Molecules do not interact with the stationary phase
  • Molecules that interact weakly elute faster (correct)
  • The interaction is solely dependent on molecular weight

What are the two primary phases involved in chromatography?

<p>Stationary phase and mobile phase (C)</p> Signup and view all the answers

Which biomolecules are usually colorless and require special detection methods in chromatography?

<p>Most biomolecules, which are generally colorless (B)</p> Signup and view all the answers

When do molecules elute from the chromatography column?

<p>When they interact poorly with the stationary phase (B)</p> Signup and view all the answers

What occurs first in the process of using chromatography for protein analysis?

<p>The lysis of cells to release proteins of interest (C)</p> Signup and view all the answers

Why is gel electrophoresis not well-suited for purifying large amounts of protein?

<p>It is primarily analytical and not designed for purification (C)</p> Signup and view all the answers

What is a potential issue when using affinity chromatography with tagged proteins?

<p>The tag may interfere with or alter the protein function. (D)</p> Signup and view all the answers

In co-immunoprecipitation, what occurs when the protein mixture interacts with the beads?

<p>Antigenic proteins interact and bind with the antibodies on the beads. (A)</p> Signup and view all the answers

What happens to the proteins that do not bind to the beads during co-immunoprecipitation?

<p>They remain in the solution and are washed away. (C)</p> Signup and view all the answers

Co-immunoprecipitation is particularly useful for identifying what?

<p>Various proteins that participate in a complex. (B)</p> Signup and view all the answers

What may occur during affinity chromatography if two proteins bind to the same target?

<p>Both proteins are retained until elution. (B)</p> Signup and view all the answers

What is the primary wavelength used for detecting proteins in chromatography?

<p>280 nm (A)</p> Signup and view all the answers

How does size-exclusion chromatography (SEC) differentiate proteins?

<p>By their physical size (A)</p> Signup and view all the answers

What happens to large proteins in size-exclusion chromatography?

<p>They elute before small proteins (C)</p> Signup and view all the answers

Which statement about the measurement of absorbance at 280 nm (A280) is true?

<p>It indicates the presence of proteins generally (B)</p> Signup and view all the answers

What is a limitation of all chromatography techniques mentioned?

<p>They can introduce impurities with the protein of interest (B)</p> Signup and view all the answers

Which of the following is true about size-exclusion chromatography compared to SDS-PAGE?

<p>Larger proteins migrate faster in SEC than in SDS-PAGE (D)</p> Signup and view all the answers

What is the function of the beads in size-exclusion chromatography?

<p>To provide a matrix for size differentiation (C)</p> Signup and view all the answers

What type of assays can be conducted on aliquots that contain protein after A280 measurements?

<p>Enzymatic and ligand-binding tests (D)</p> Signup and view all the answers

What primarily determines how quickly proteins elute in size-exclusion chromatography?

<p>The size of the proteins (C)</p> Signup and view all the answers

Which of the following statements is true about the separation of proteins in size-exclusion chromatography?

<p>Most proteins resolved are of intermediate size. (A)</p> Signup and view all the answers

What is the main driving force for protein migration in size-exclusion chromatography?

<p>Gravity or pump action (B)</p> Signup and view all the answers

How does size-exclusion chromatography affect the protein structure during separation?

<p>It preserves the proteins' secondary and tertiary structures. (D)</p> Signup and view all the answers

In ion-exchange chromatography, how do positively charged proteins interact with the stationary phase?

<p>They bind to negatively charged beads. (B)</p> Signup and view all the answers

Why are proteins that differ in size by only a few amino acids unlikely to be separated well by size-exclusion chromatography?

<p>Their sizes are too similar to create a distinction. (C)</p> Signup and view all the answers

Which characteristic of proteins does ion-exchange chromatography primarily exploit?

<p>Their electrical charge (C)</p> Signup and view all the answers

What happens to proteins that are too large to fit into the pores during size-exclusion chromatography?

<p>They only travel through the void volume. (B)</p> Signup and view all the answers

What is the primary advantage of affinity chromatography over size-exclusion and ion-exchange chromatography?

<p>It can select for a specific protein of interest. (C)</p> Signup and view all the answers

What role do tags play in affinity chromatography?

<p>They are necessary for the elution of proteins. (B)</p> Signup and view all the answers

Which of the following statements about affinity chromatography is true?

<p>Each protein requires a unique column with specific beads. (D)</p> Signup and view all the answers

How can proteins tagged with Myc tags be eluted from an affinity column?

<p>By adding short peptides expressing the Myc sequence. (C)</p> Signup and view all the answers

What is a potential limitation of using multiple affinity chromatography columns for different proteins?

<p>Each column requires specific tags and may not be cost-effective. (A)</p> Signup and view all the answers

Which method is used to disrupt protein-bead interactions in affinity chromatography?

<p>Addition of a competitive inhibitor. (A)</p> Signup and view all the answers

What is the function of polyhistidine tags in protein purification?

<p>To facilitate binding to nickel columns for purification. (D)</p> Signup and view all the answers

What distinguishes affinity chromatography from other chromatographic techniques?

<p>It selectively binds particular proteins through specific interactions. (C)</p> Signup and view all the answers

What is the main principle behind cation-exchange chromatography?

<p>Cations from added salt compete with cationic proteins for binding. (C)</p> Signup and view all the answers

How is the charge of a protein related to its isoelectric point (pl) and the pH of the buffer?

<p>Proteins with pl below the pH of the buffer have a net positive charge. (C)</p> Signup and view all the answers

Which statement regarding elution in ion-exchange chromatography is correct?

<p>The order of elution can be predicted from the isoelectric points of proteins. (A)</p> Signup and view all the answers

How do uncharged proteins behave during ion-exchange chromatography?

<p>They elute quickly, even without added salt. (C)</p> Signup and view all the answers

What role do antibodies play in affinity chromatography?

<p>They are linked to beads to bind specific target proteins. (B)</p> Signup and view all the answers

In cation-exchange chromatography, what happens at high salt concentrations?

<p>Cations from salt outcompete the bound cationic proteins. (C)</p> Signup and view all the answers

What characteristic describes the binding of proteins with larger charge magnitudes in ion-exchange chromatography?

<p>They bind more tightly than proteins with smaller charges. (B)</p> Signup and view all the answers

What occurs when proteins with the same charge as the beads are in the column?

<p>They are repelled and elute faster than uncharged proteins. (C)</p> Signup and view all the answers

Flashcards

Chromatography

A laboratory technique used to separate molecules based on their physical properties.

Stationary Phase

The solid phase in chromatography, typically consisting of small beads or a gel.

Mobile Phase

The liquid phase in chromatography, which carries the molecules through the stationary phase.

Affinity

The interaction between molecules in the mobile phase and the stationary phase.

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Elution

The process of molecules exiting the chromatography column.

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Lysis

The process of breaking open cells to release their contents.

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Gel Electrophoresis

The separation of molecules based on their size and charge.

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Blotting

A technique that transfers molecules from a gel to a membrane.

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Size Exclusion Chromatography (SEC)

A technique used to separate proteins based on their size, also known as gel filtration chromatography.

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SEC Bead Cutoff

The specific size of a protein that can pass through a SEC column's beads (pores). Different beads have different cutoffs for different sized proteins.

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Aliquot

The process of collecting small volumes of buffer exiting a chromatography column. Often used to monitor and analyze the elution process.

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A280 (Absorbance at 280 nm)

The measurement of the absorbance of 280 nm light, used to detect proteins in a solution or elution buffer.

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Elution Order in SEC

In SEC, larger proteins elute first because they cannot enter the beads' pores, taking a shorter path through the column.

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Specific Protein Activity Assay

A method to confirm the presence of a specific protein of interest within a collected aliquot from a column.

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Multi-Step Chromatography

The use of multiple chromatography techniques in sequence to increase the purification and remove impurities from a protein sample.

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Affinity Chromatography

A technique that utilizes the separation of proteins based on their binding affinity to a stationary phase.

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Void Volume

A chamber within the SEC column that larger proteins flow through, avoiding the pores of the stationary beads. This volume only includes the space between the beads.

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Total Volume

The volume that small proteins traverse, including both the spaces between the beads and the volume within the pores of the stationary beads.

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Force in SEC

SEC relies on gravity or a pump to move the mobile phase carrying the proteins through the column.

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Force in Electrophoresis

Electrophoresis uses an electric current to force proteins through a gel matrix.

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Protein Denaturation in SEC

SEC does not typically denature proteins, preserving their biological activity.

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Ion Binding

The process of proteins binding to charged beads in ion-exchange chromatography.

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Ion-Exchange Chromatography

Ion-exchange chromatography separates proteins based on their charge.

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Isoelectric point (pI)

The pH at which a protein has no net charge.

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Elution with salt

The process of adding salt to the mobile phase in ion-exchange chromatography to release bound proteins.

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Cation-exchange chromatography

The process of separating proteins based on their charge, using beads with opposite charges.

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Anionic proteins

Proteins with a net negative charge at a given pH.

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Cationic proteins

Proteins with a net positive charge at a given pH.

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Ligand

A substance that binds to the stationary phase in affinity chromatography, targeting a specific protein.

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Competitive Inhibitor

An added substance with high affinity for the stationary phase that disrupts the binding of the target protein, allowing its elution.

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Tag

A sequence of amino acids added to a protein, allowing specific targeting and purification.

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Myc Tag

A type of tag that binds to specific antibodies.

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Polyhistidine Tag

A type of tag that binds to nickel.

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Co-Immunoprecipitation (coIP)

A technique that uses antibodies attached to beads to isolate proteins from a mixture, allowing identification of proteins that interact with the protein of interest.

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Co-immunoprecipitated proteins

Proteins that bind to the target protein through interaction, not directly to the antibody, and become trapped along with the target protein during co-immunoprecipitation.

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CoIP: Identifying Protein Interactions

A technique used to determine the protein binding partners of a target protein, utilizing antibodies and beads to isolate and identify interacting proteins.

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Immunoprecipitation

The process of removing proteins from a solution by using antibodies attached to beads, similar to fishing with a specific bait.

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Supernatant

The liquid remaining after a protein has been separated from the solution using beads and antibodies, leaving behind the target protein and any interacting proteins.

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Study Notes

Chromatography Introduction

  • Chromatography is a technique used to separate proteins and other biomolecules for analysis or purification.
  • Gel electrophoresis and blotting techniques are primarily for protein analysis, not purification.
  • Purifying proteins often requires expression in large quantities and cell lysis to release proteins of interest.
  • Chromatography separates mixtures based on different physical properties.

Principles of Chromatography

  • Chromatography involves a stationary phase (beads/gel) and a mobile phase (solution).
  • Molecules in the mobile phase interact differently with the stationary phase, causing them to move at varying speeds.
  • Molecules interacting strongly move slowly, while those interacting weakly move quickly.

Column Chromatography

  • Separates molecules in a mobile phase based on interaction strength with the stationary phase.
  • Molecules interacting strongly move slower through the column.
  • Molecules interacting weakly elute (exit) faster.

Size Exclusion Chromatography (SEC)

  • Also called gel filtration chromatography.
  • Proteins of different sizes move through the column at varying rates.
  • Smaller proteins enter the pores of the gel beads, increasing their interaction time and slower movement.
  • Larger proteins are excluded and move more quickly.
  • Large proteins elute first, contrasting with electrophoresis.

Detection of Proteins

  • Colored proteins can be detected visually.
  • Colorless proteins use spectrophotometers.
  • Absorbance at 280 nm (A280) helps detect proteins.
  • Protein activity assays confirm if the solution contains the protein of interest.

Other Chromatography Techniques

  • Chromatography is not perfect, some impurities are often part of the purified protein sample.
  • The application of multiple chromatography techniques can increase purity to extract desired proteins.

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Biochemistry 14.3 Chromatography PDF

Description

This quiz delves into the fundamental concepts of chromatography in biochemical analysis. Explore the interactions between biomolecules and the stationary phase, the phases involved, and specific challenges related to protein purification. Test your understanding of these essential techniques used in biochemical laboratories.

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