Podcast
Questions and Answers
What are the primary factors that purification techniques focus on?
What are the primary factors that purification techniques focus on?
Size and charge
Which of the following methods are used in homogenization?
Which of the following methods are used in homogenization?
What is the purpose of differential centrifugation in protein purification?
What is the purpose of differential centrifugation in protein purification?
Separate cellular components based on their size and density.
Which of the following techniques is used to increase protein solubility?
Which of the following techniques is used to increase protein solubility?
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Which of the following techniques is used to decrease protein solubility and precipitate proteins?
Which of the following techniques is used to decrease protein solubility and precipitate proteins?
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What is the most common reagent used in salting-out?
What is the most common reagent used in salting-out?
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Salting-out is considered a very precise protein purification technique.
Salting-out is considered a very precise protein purification technique.
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What is the principle behind dialysis?
What is the principle behind dialysis?
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What is meant by 'MW cut-off' in dialysis?
What is meant by 'MW cut-off' in dialysis?
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Which technique involves separating molecules based on their affinity for a stationary phase?
Which technique involves separating molecules based on their affinity for a stationary phase?
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What are the two phases involved in chromatography?
What are the two phases involved in chromatography?
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Column chromatography is only used for colorful proteins.
Column chromatography is only used for colorful proteins.
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What are the two main steps involved in column chromatography?
What are the two main steps involved in column chromatography?
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What is the basis of separation in size-exclusion chromatography?
What is the basis of separation in size-exclusion chromatography?
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What are the two main types of polymers used in size-exclusion chromatography?
What are the two main types of polymers used in size-exclusion chromatography?
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What is the key factor determining separation in size-exclusion chromatography?
What is the key factor determining separation in size-exclusion chromatography?
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Size-exclusion chromatography can provide estimates of the molecular weight of proteins.
Size-exclusion chromatography can provide estimates of the molecular weight of proteins.
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What is the principle behind ion-exchange chromatography?
What is the principle behind ion-exchange chromatography?
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Ion-exchange chromatography is highly specific for one particular protein.
Ion-exchange chromatography is highly specific for one particular protein.
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What are the typical counter-ions that bind to a cation-exchange resin?
What are the typical counter-ions that bind to a cation-exchange resin?
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Study Notes
Protein Purification and Characterization Techniques
- Techniques focus on size and charge
- Homogenization is the initial stage (grinding, homogenizers, sonication, etc.)
- Differential centrifugation separates components based on size/density (unbroken cells, nuclei, mitochondria, ribosomes, etc.)
Salting In & Out
- Salts impact protein solubility by varying charged groups/ionic strength
- Ammonium sulfate is a common reagent
- This technique can be crucial but results can be crude. Protein solubility changes with salt concentration.
Dialysis
- Purification technique based on diffusion
- Separates molecules by molecular weight cut-off
- Compares pure vs. crude samples
Column Chromatography
- Technique uses stationary and mobile phases
- Separates components based on affinity to each phase
- Includes washing/elution to separate compounds
Gel-filtration Chromatography
- Separates based on molecular weight (MW)
- Stationary phase is cross-linked gel particles (dextran, agarose, polyacrylamide)
- Separation based on the size of molecules. Larger molecules elute first.
Molecular-sieve chromatography
- Separates molecules by size
- Larger molecules elute first, smaller molecules take longer.
Ion-exchange Chromatography
- Separates based on charge (cation or anion exchangers)
- Resin bound to ions
- Elution occurs with higher salt concentration
- Different resins for different functionalities (e.g., CM cellulose, DEAE cellulose)
Isoelectric Focusing
- Proteins have different isoelectric points
- Separates components based on pH gradient/electric field
- 2D gel electrophoresis helps analyze proteins in more detail.
Agarose or PAGE?
- Agarose for nucleic acids, PAGE for proteins.
- SDS-PAGE denatures proteins
- Native PAGE preserves native protein conformation.
Immunoassays - ELISA
- Detects/quantifies substances (peptides, proteins, antibodies, hormones)
- Commonly used in 96-well plates
- Rapid, convenient & sensitive method
- Applications in screening, allergen detection, and hormonal testing.
Protein Sequencing
- Determining the amino acid sequence of a protein.
- Edman degradation is a crucial technique: it cleaves and identifies N-terminal amino acids step-by-step.
- Needs protein hydrolysis and separation by ion-exchange chromatography (or HPLC)
Cleavage Methods
- Chemical digestion, Endopeptidases, and Exopeptidases.
Chemical Digestion
- Using cyanogen bromide (CNBr) for specific cleavage at methionine residues.
Endopeptidases
- Cleave at specific sites within the primary protein sequence
Exopeptidases
- Cleave amino acids from the ends of the peptide (N-terminus or C-terminus)
Protein Sequencing - Prediction
- If the gene sequence is known, sequencing proteins is easy.
- Unknown? Steps like complementary RNA, mRNA isolation, PCR, and gene sequencing are used.
Determination of 3° Structure
- X-ray crystallography uses crystals and x-rays to determine molecular coordinates.
- 2D Nuclear Magnetic Resonance (NMR) can be used in aqueous solutions.
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Description
This quiz covers various methods for protein purification and characterization, including techniques like salting in and out, dialysis, column chromatography, and gel-filtration chromatography. Test your knowledge on how these methods utilize size and charge to achieve efficient purification.
