Principle: Separation of protein molecules based on size and their ability to enter the pores of the gel.
2. Ion-Exchange Chromatography
Process: Bound molecules are eluted by increasing ionic strength (adding salt) or changing pH of the elution buffer.
Separation Based On: Charge properties and reversible binding to charged resin particles.
3. Affinity Chromatography
Process: Proteins are separated based on affinity for specific ligands and eluted by adding free ligands to the elution buffer.
Electrophoretic Analysis of Proteins
Proteins can be separated and identified via gel electrophoresis based on size (MW) and/or net charge (pI).
Electrophoresis Explained: The separation of charged molecules (DNA, Proteins) under electric field influence through gel pores.
Gel Electrophoresis Types
Polyacrylamide Gel Electrophoresis (PAGE): Most common medium; acrylamide and bisacrylamide form the gel.
SDS-PAGE (Sodium Dodecyl Sulfate-PAGE)
Function: Accurately determines polypeptide size under denaturing conditions.
Operation: SDS disrupts protein structures, providing them with an overall negative charge, allowing their separation based only on size.
Reducing Agents: DTT or β-mercaptoethanol cleave disulfide bonds to separate protein subunits.
Electrophoretic Mobility
Relationship to MW: Electrophoretic mobility (Rf) is inversely proportional to the logarithm of the protein's MW.
Formula: ( Rf = \frac{x}{G} ) where x is the distance traveled by the protein, and G is the distance traveled by the front.
Isoelectric Focusing
Method: For separating proteins based on pI, proteins migrate through a gel with a pH gradient until they reach the point where their charge is neutral (pI).
Two-Dimensional Electrophoresis
Combination of Techniques: Allows for enhanced resolution in separating proteins, facilitating the analysis of expression levels and identification for diagnostic purposes.
Evaluation of Protein Purification
Monitor qualitatively using SDS-PAGE.
Monitor quantitatively using total protein, total activity, specific activity, yield, and purification level after each step.
Example 2: Purification of a Protein Without Enzymatic Activity (Estrogen Receptor)
Detection Method: Monitoring distinct binding properties (e.g., receptor-estradiol complex detection) using techniques like zonal centrifugation.
Antibody-Related Techniques
Antibody Production
Antibodies can be raised against specific antigens by injecting the protein or parts of it into animals and isolating their serum.
Monoclonal Antibodies
Created by fusing antibody-producing cells with cancer cells (e.g., multiple myeloma).
Clinical Uses: For detecting proteins and viruses and as therapeutic agents.
Immunological Techniques in Protein Analysis
Immunoprecipitation
A method where the target protein is “fished” out using a monoclonal antibody.
ELISA (Enzyme-Linked Immunosorbent Assay)
For detection and quantification of proteins.
Examples include HIV tests and pregnancy tests.
Western Blotting (Immunoblotting)
Process: Electrotransfer proteins to a membrane, followed by incubation with specific antibodies.
Used for diagnostic purposes, such as viral protein detection and identification of oligoclonal bands in cerebrospinal fluid for diagnosing multiple sclerosis.
Detection of Antigens
Methods include rapid tests (lateral flow tests, dipsticks) for various antigens such as those for COVID-19.
Determination of Amino Acid Sequence
Determination Techniques
Amino Acid Composition: E.g., with Ala-Gly-Asp-Phe-Arg-Gly subjected to hydrolysis.
Ion Exchange Chromatography: To separate amino acids based on charge, eluating with a pH gradient buffer.
Edman Degradation: To determine the amino terminal residue, allowing the sequence for peptides up to 50 amino acids to be determined.
Protein Cleavage for Sequencing
Polypeptides can be cleaved at specific sites to facilitate sequencing; sequences can be determined through overlapping peptide analysis.
Mass Spectrometry (MS)
Allows for precise determination of protein mass, with techniques like MALDI-TOF facilitating identification based on peptide fingerprints.
Tandem Mass Spectrometry
For amino acid sequencing of peptides, enabling protein gene cloning through DNA sequence determination and translation according to the genetic code.
Importance of Amino Acid Sequence
Identifies function, enabling comparison and classification of proteins.
Allows evolutionary comparisons amongst species.
Facilitates detection of mutations leading to disease at the molecular level.
Summary of Protein Analysis Methods
Techniques and methodologies detailed throughout ensure robust understanding and practical application in protein analysis critical in both research and clinical settings.
Revision Questions
An enzyme that binds strongly to an ion exchange chromatography gel and elutes slowly:
A. has the same charge as the resin at the pH of the elution solution.
B. has a size larger than the gel pore size.
C. can be eluted by adding its substrate to the elution solution.
D. can be eluted by adding salt to the elution solution.
E. can be eluted by reducing the ionic strength of the elution solution.
The Western immunoblotting method includes all the following except: