This free course comprises two interactive modules that were developed to provide a succinct, contextual overview of protein sample isolation using total protein extraction and subcellular fractionation. The module covers methods for purifying whole cell lysates and describes strategies for protein cleanup procedures.
Overview
The goal of protein sample preparation is to generate quality protein samples that maximize the chance of a successful downstream application. This course provides information about how to select the best workflow as it relates to these variables: sample type, sample volume, protein characteristics, protein yield required, and intended downstream application. At the end of each course, you will have the opportunity to apply this information about common protein sample preparation techniques and methods by completing the associated practical application exercises.
The topics covered in the Protein sample preparation: Techniques and applications eLearning course are:
Part 1: Total protein extraction; cell disruption methods: (reagent-based and physical); subcellular fractionation; sample preparation for downstream assays such as:
- Western blotting
- Enzyme-linked immunosorbent assays (ELISA)
- Immunoprecipitation
- Mass spectrometry
Part 2: Hydrophobic interaction chromatography (HIC); size exclusion chromatography (SEC); ion exchange chromatography (IEX); affinity chromatography (AC); protein cleanup methods:
- Desalting
- Buffer exchange
- Dialysis
Each module is interactive, and you will have opportunities to test your knowledge along the way. Each module takes approximately 20 minutes to view. The tabs below provide more detailed descriptions and the learning objectives for each module.
Protein Extraction
This is the first section of this four -part series and describes common methods for protein sample preparation.
Upon completion of this module, you will be able to:
- Understand the basic work flow of protein sample preparation.
- Use protein sample preparation and purification techniques in a practical setting.
- Develop specific protocols for protein extraction from cells and tissues.
Protein preparation decision tree. This illustration depicts many of the factors involved in selecting a particular protein isolation strategy.
Technical content excerpt
The first step in protein sample preparation is the identification of a suitable protein source for sample collection. Proteins can be obtained from their native source material such as mammalian cell cultures, tissues or body fluids, or from their overexpression in a model system such as bacteria, yeast, or mammalian cells. Hybridoma cells are a source for monoclonal antibodies. Plant cells are a source of protein in agricultural biotechnology.
Sample collection removes the cells or tissue samples from their natural environment, leading to a disturbance in cellular homeostasis. This activates stress responses which in turn lead to an increased activity of various proteases and enzymes responsible for post-translational modifications that contribute to the degradation or alteration of proteins in a sample during or after sample collection.
Various strategies can be employed at the time of sample collection to limit the degradation of the proteome, including immediate freezing of the sample in liquid nitrogen, chemical and/or temperature-induced denaturation of protein modifying enzymes, the addition of protective or stabilizing compounds such as reducing agents, enzyme inhibitors, etc, stabilization or inactivation of proteins by precipitation and working quickly and keeping samples cold during processing.
Source of specimen or sample type. Suitable protein sources for sample collection may include native sources derived from mammalian cell cultures, tissues or body fluids. Alternatively proteins may be over expressed in model systems such as yeast, bacteria, insect or mammalian cells.
Considerations in protein sample preparation. Removing cells or tissue samples from their natural environment causes disturbances in cellular homeostasis. Samples must be protected from degradation by various proteases and enzymes during protein isolation procedures.
Protein purification
This is the second section of this four-part series and describes various approaches for protein purification.
Upon completion of this module, you will be able to:
- Identify and understand the differences between major methods and strategies for selecting the correct protein purification method.
- Understand how to choose the correct protein purification resin.
- Select the appropriate protein cleanup strategy.
Protein separation by column chromatography. Subsequent to removal of the gross contaminants from the crude protein extract, the remaining protein components may be resolved, and a specific protein may be purified. One of the most ubiquitous techniques for protein separation involves the use of column chromatography. This form of chromatography utilizes chemical and biological properties of the protein for its purification, and achieves high resolution separation.
Technical content excerpt
Protein purification is separating the protein of interest in its desired form from all other proteins or from product and process related impurities.
Protein purification is one of the most important parts of protein research, and is a prerequisite when studying unique characteristics of proteins such as the size, charge, shape, and function. Downstream techniques for analyzing purified proteins include crystallography or, sequencing using mass spectrometry.
Protein purification begins with preparation and extraction of the protein. This is followed by a capture phase in which the protein is isolated, concentrated, and stabilized. Next, impurities (such as other proteins, nucleic acids, endotoxins, and viruses) are removed in a wash phase. Lastly, a high level of purification is achieved in an elution phase—this removes any remaining trace impurities or other contaminants.
Different degrees of protein purification may be required, depending, for instance, on downstream analyses to be performed. The steps outlined above may be omitted or repeated in order to achieve the desired level of protein purification.
Click image to enlarge
Workflow for the purification of recombinant protein from BL21 competent E. coli. This schematic shows steps involved in a protein purification strategy that employs immobilized metal affinity chromatography (IMAC) beads.
Protein purification workflow. The steps outlined above may be omitted or repeated in order to achieve the desired level of protein purification.
Techniques and applications—Part 1
This is the third section of this four-part series designed as a practical application to provide opportunities to integrate the information outlined in the narrated portion of the eLearning course.
Protein sample preparation. The workflow begins with cell lysis, and the steps involved in sample preparation are designed to move the protein containing fraction from the initial biological source into a homogeneous solution, while preserving the in vivo state of the protein. Obtaining high quality lysates is critical for the successful outcome of downstream applications.
Technical content excerpt
Cell lysis and protein extraction
These exercises guide the learner through different sample preparation workflows required for obtaining high quality lysates needed for successful western blot experiments.
Protein extraction techniques
These exercises guide the learner through different sample preparation techniques designed to extract protein from mammalian cells or tissues.
Techniques and applications—Part 2
This is the fourth section of this four-part series designed as a practical application to provide opportunities to integrate the information outlined in the narrated portion of the eLearning course.
Ion exchange chromatography (IEX). This technique is used to separate proteins based on differences in surface charge and provides medium to high resolution separation and has a high sample loading capacity. IEX separates proteins based on a reversible interaction between a charged protein and an oppositely charged chromatography medium, also called a resin.
Technical content excerpt
Protein purification strategies
These exercises guide the learner through different protein purification workflows required for obtaining high quality lysates needed for successful down-stream applications, such as mass spectrometry.
These exercises guide the learner through different protein purification techniques designed to isolate native or recombinant affinity tagged proteins.
Protein Extraction
This is the first section of this four -part series and describes common methods for protein sample preparation.
Upon completion of this module, you will be able to:
- Understand the basic work flow of protein sample preparation.
- Use protein sample preparation and purification techniques in a practical setting.
- Develop specific protocols for protein extraction from cells and tissues.
Protein preparation decision tree. This illustration depicts many of the factors involved in selecting a particular protein isolation strategy.
Technical content excerpt
The first step in protein sample preparation is the identification of a suitable protein source for sample collection. Proteins can be obtained from their native source material such as mammalian cell cultures, tissues or body fluids, or from their overexpression in a model system such as bacteria, yeast, or mammalian cells. Hybridoma cells are a source for monoclonal antibodies. Plant cells are a source of protein in agricultural biotechnology.
Sample collection removes the cells or tissue samples from their natural environment, leading to a disturbance in cellular homeostasis. This activates stress responses which in turn lead to an increased activity of various proteases and enzymes responsible for post-translational modifications that contribute to the degradation or alteration of proteins in a sample during or after sample collection.
Various strategies can be employed at the time of sample collection to limit the degradation of the proteome, including immediate freezing of the sample in liquid nitrogen, chemical and/or temperature-induced denaturation of protein modifying enzymes, the addition of protective or stabilizing compounds such as reducing agents, enzyme inhibitors, etc, stabilization or inactivation of proteins by precipitation and working quickly and keeping samples cold during processing.
Source of specimen or sample type. Suitable protein sources for sample collection may include native sources derived from mammalian cell cultures, tissues or body fluids. Alternatively proteins may be over expressed in model systems such as yeast, bacteria, insect or mammalian cells.
Considerations in protein sample preparation. Removing cells or tissue samples from their natural environment causes disturbances in cellular homeostasis. Samples must be protected from degradation by various proteases and enzymes during protein isolation procedures.
Protein purification
This is the second section of this four-part series and describes various approaches for protein purification.
Upon completion of this module, you will be able to:
- Identify and understand the differences between major methods and strategies for selecting the correct protein purification method.
- Understand how to choose the correct protein purification resin.
- Select the appropriate protein cleanup strategy.
Protein separation by column chromatography. Subsequent to removal of the gross contaminants from the crude protein extract, the remaining protein components may be resolved, and a specific protein may be purified. One of the most ubiquitous techniques for protein separation involves the use of column chromatography. This form of chromatography utilizes chemical and biological properties of the protein for its purification, and achieves high resolution separation.
Technical content excerpt
Protein purification is separating the protein of interest in its desired form from all other proteins or from product and process related impurities.
Protein purification is one of the most important parts of protein research, and is a prerequisite when studying unique characteristics of proteins such as the size, charge, shape, and function. Downstream techniques for analyzing purified proteins include crystallography or, sequencing using mass spectrometry.
Protein purification begins with preparation and extraction of the protein. This is followed by a capture phase in which the protein is isolated, concentrated, and stabilized. Next, impurities (such as other proteins, nucleic acids, endotoxins, and viruses) are removed in a wash phase. Lastly, a high level of purification is achieved in an elution phase—this removes any remaining trace impurities or other contaminants.
Different degrees of protein purification may be required, depending, for instance, on downstream analyses to be performed. The steps outlined above may be omitted or repeated in order to achieve the desired level of protein purification.
Click image to enlarge
Workflow for the purification of recombinant protein from BL21 competent E. coli. This schematic shows steps involved in a protein purification strategy that employs immobilized metal affinity chromatography (IMAC) beads.
Protein purification workflow. The steps outlined above may be omitted or repeated in order to achieve the desired level of protein purification.
Techniques and applications—Part 1
This is the third section of this four-part series designed as a practical application to provide opportunities to integrate the information outlined in the narrated portion of the eLearning course.
Protein sample preparation. The workflow begins with cell lysis, and the steps involved in sample preparation are designed to move the protein containing fraction from the initial biological source into a homogeneous solution, while preserving the in vivo state of the protein. Obtaining high quality lysates is critical for the successful outcome of downstream applications.
Technical content excerpt
Cell lysis and protein extraction
These exercises guide the learner through different sample preparation workflows required for obtaining high quality lysates needed for successful western blot experiments.
Protein extraction techniques
These exercises guide the learner through different sample preparation techniques designed to extract protein from mammalian cells or tissues.
Techniques and applications—Part 2
This is the fourth section of this four-part series designed as a practical application to provide opportunities to integrate the information outlined in the narrated portion of the eLearning course.
Ion exchange chromatography (IEX). This technique is used to separate proteins based on differences in surface charge and provides medium to high resolution separation and has a high sample loading capacity. IEX separates proteins based on a reversible interaction between a charged protein and an oppositely charged chromatography medium, also called a resin.
Technical content excerpt
Protein purification strategies
These exercises guide the learner through different protein purification workflows required for obtaining high quality lysates needed for successful down-stream applications, such as mass spectrometry.
These exercises guide the learner through different protein purification techniques designed to isolate native or recombinant affinity tagged proteins.
For Research Use Only. Not for use in diagnostic procedures.