Protein detection
Page 1: Introduction to Protein Work
Experimental Tools in Cell Biology
Working with proteins involves several steps:
Extraction of proteins
Separation by gel electrophoresis
Purification of specific proteins
Detection of proteins
Proteins can be hydrophilic or hydrophobic and exhibit diverse properties (solubility, shape, charge).
Extraction can be challenging, and purification varies for each protein.
Page 2: Protein Purification Techniques
Overview of Purification Techniques
Extraction can yield partial purification.
Considerations in purification:
Soluble vs. membrane spanning proteins
Organelle isolation
Heat stability
Further purification techniques involve chromatography, which includes:
Ion Exchange (charge separation)
Gel Filtration (size fractionation)
Hydrophobic Interaction
Affinity Chromatography (specific binding)
Page 3: Ion-Exchange Chromatography Basics
Mechanism of Ion-Exchange Chromatography
Proteins with opposite net charge bind to a charged stationary matrix.
Proteins with the same charge pass through unbound.
Elution involves a salt gradient, where the weakest charged proteins elute first, allowing for the collection of similar ionic properties in fractions.
Page 4: Common First Step in Ion-Exchange Chromatography
The column matrix is positively or negatively charged.
The binding concentrates proteins from diluted extracts.
Elution using a salt gradient results in concentrated peaks of different protein classes.
Page 5: Anion Exchange Chromatography
Focus on Negatively Charged Proteins
Anion exchange chromatography binds negatively charged proteins.
Wash steps involve using a mix of amino acids and buffer.
Page 6: Elution of Negatively Charged Proteins
Elution Process
Negatively charged proteins are eluted using salt as a competitor.
Mixture of amino acids and salt gradient facilitates elution.
Page 7: Gradient Mixer in Ion-Exchange Chromatography
Gradually increases salt concentration from 0 mM to 500 mM.
Utilizes 1M NaCl in buffer for the elution process.
Page 8: Ion-Exchange Chromatography Procedure
Steps and Parameters
Elution involves using salt to wash away bound proteins, followed by high salt wash and re-equilibration.
Samples are concentrated and fractions collected based on elution volume.
Page 9: De-salting Techniques
Removal of Salt After Ion Exchange
De-salting can be performed using dialysis (semi-permeable membranes).
Allows for the separation of salt from concentrated solutions.
Page 10: De-salting with Gel-Filtration Columns
Advantage of Gel-Filtration for Desalting
Offers faster and more efficient desalting.
Operates on the principle of size exclusion.
Page 11: Gel Filtration Overview
Size Exclusion Chromatography
Requires concentrated and clean samples for effective separation.
Small proteins are slowed down while larger proteins progress more rapidly.
Page 12: Further Purification with Gel Filtration
Fraction Collection
Each fraction contains proteins with similar features, progressing towards purifying the target protein.
Allows for further separation based on different properties such as hydrophobicity.
Page 13: Affinity Chromatography
Binding Specificity
Only proteins with affinity for the matrix will remain bound.
Other proteins will elute regardless of their properties.
Contaminants can be removed post-binding through other separation methods.
Page 14: Practical Column Used
Column Description
Details about the gel-filtration column to be utilized for sample migration.
Page 15: Protein Gels Overview
Monitoring Protein Purification
Protein gels essential for tracking the progress of purification.
Page 16: Making a Polyacrylamide Gel
Gel Preparation Process
Assemble glass plates, pour gel, insert comb, and polymerize.
Prepare for electrophoresis.
Page 17: Real Lab Practicals
Practical Example
Scaffold assembly for polyacrylamide gel ready for casting.
Page 18: SDS-PAGE Technique
Size-Based Separation
Smaller proteins migrate faster than larger in denaturing gels (SDS-PAGE), allowing size-based separation after denaturation.
Page 19: Coomassie Staining
Detection of Proteins
Coomassie blue staining shows protein bands, indicative of abundance after size-based separation.
Page 20: Electroblotting Technique
Protein Transfer
Proteins must be transferred to a nitrocellulose membrane for immunodetection.
Page 21: Immunodetection Steps
Procedure for Antibody Incubation
Block membrane with milk proteins.
Add specific antibody and incubate.
Wash away unbound antibodies.
Add secondary antibody for signal detection.
Wash and visualize the signal.
Page 22: Wash Steps
Detailed Wash Procedure
Series of washes are crucial for specificity and reducing background noise during detection.
Page 23: Comparison of Detection Methods
Coomassie vs. Immunodetection
Coomassie stains all proteins while immunodetection targets specific antigens, allowing comparison of abundance and specificity.
Page 24: Understanding Antibodies
Primary and Secondary Antibodies
Primary antibodies target the specific protein; secondary antibodies amplify the signal.
Use of secondary antibodies aids in detection and reduces labelling effort.
Page 25: Generating Antibodies
Need for Antibodies
Purify protein.
Generate antibodies (polyclonal in rabbits or monoclonal from hybridomas for targeting proteins).