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5 Steps to Fundamental Protein Preparation |
The goal of protein preparation is to generate quality protein samples that maximize the chance of a successful downstream analysis (e.g., western blotting, ELISA, immunoprecipitation, mass spectrometry). Because proteins are diverse in both structure and in function, there are often challenges with balancing efficient extraction and maintaining protein function required for the downstream analysis. This balance is crucial for studies to examine biological phenomena, and success is dependent on the methods and reagents required for protein preparation.
How to prepare protein samples
Preparing protein samples is dependent on multiple factors. The objective of protein sample preparation is to create a homogeneous solution while preserving the protein's in vivo state for the best results in downstream experimental applications. Factors determining the protein preparation products and methods used include sample type or source, protein location, desired yield, and downstream application.
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Figure 1. 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.
Click image to enlarge
Figure 2. 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.
Figue 3. Protein preparation decision tree. This illustration depicts many of the factors involved in selecting a particular protein isolation strategy.
Protein sample prep steps
The five steps of protein sample preparation to help obtain high-quality protein samples include:
- Protein extraction
- Protein preservation and stabilization
- Protein clean-up and concentration
- Protein quantitation
- Protein detection
View the steps of the workflow below to find recommendations and related topics.
Extract your target protein from the sample
The first step in protein analysis is cellular extraction, which requires liberation of protein from the sample source. Whether using mechanical or detergent-based extraction methods, this process inevitably lyses the cell and contributes to the degradation or destabilization of proteins. Therefore, the quality of the data obtained from protein samples directly depends upon the integrity of the protein during the extraction process. For example, some extraction methods may be efficient at cell lysis and solubilization of cell contents but are protein-denaturing, thereby preventing detection and analysis of native protein interactions.
Sample types
Cultured mammalian cells, mammalian tissues, and primary cells are frequently used as sources of physiologically relevant endogenous proteins (including post-translational modifications) as well as overexpression systems (transient or stable). When extracting protein from mammalian tissues, some gentle means of enzymatic and/or mechanical disruption is required to help separate cells from the more complex tissue matrix. For cultured mammalian cells and primary cells that only have a plasma membrane separating the cell contents from the environment, reagents containing detergents can disrupt the protein-lipid membrane bilayer, making total protein extraction relatively easy. Other organisms that are commonly studied or used for recombinant protein expression systems include bacteria, yeast, and plants. These cell types contain cell walls that require additional enzymatic or mechanical disruption to efficiently release their protein content. However, detergent-based solutions have been developed to effectively extract and solubilize protein from these cells without the need for mechanical disruption, which is especially important when processing higher sample numbers or when automating extraction and purification protocols.
Sub-cellular fractionation
For many studies, generating whole cell lysates is an easy, straightforward way to prepare a soluble protein sample for direct detection or for further purification or fractionation. However, the yield or enrichment of a specific protein can be improved significantly if the cell is fractionated into different compartments or organelles before protein extraction.
Mechanical lysis usually disrupts all cellular compartments, thereby making it difficult to isolate specific cellular fractions. However, by carefully optimizing reagents, stepwise differential detergent procedures have been developed to separate nuclear, cytosolic, and membrane protein fractions. With this strategic approach, hydrophobic membrane proteins can be solubilized and separated from the hydrophilic proteins, and intact nuclei, mitochondria, and other organelles can be isolated for direct study or separate protein extraction.
Pro tip
Analyze a sample of the solubilized protein and the insoluble fractions by SDS-PAGE to determine the efficiency of the protein extraction method used.
Products and resources for protein extraction
Preserve your target protein
Cell lysis disrupts cell membranes and organelles, resulting in proteolytic activity that can reduce protein yield and function. To prevent degradation of extracted proteins and maintain their activity, protease and phosphatase inhibitors are frequently added to lysis reagents.
Protease inhibitors function by binding to protease active sites. Due to the differences in the proteolytic mechanisms, no single compound can effectively inhibit all proteases, and therefore, a mixture or cocktail of several different inhibitor compounds is needed to help ensure that protein extracts do not degrade before downstream analyses. Typical cocktails include small molecule inhibitors of serine, cysteine, and aspartic acid proteases as well as aminopeptidases and metalloproteases. While some inhibitors are irreversible, many are reversible and require their continued presence in the crude sample until further purification removes the threat of proteolytic activity.
Likewise, phosphatases vary so an effective phosphatase cocktail containing inhibitors for serine, threonine, tyrosine, acidic, and alkaline phosphatases is recommended to preserve fragile phosphorylation post-translational modifications.
Most researchers use a mixture of several different inhibitor compounds to help ensure the protein extracts do not degrade before analysis of target interest. Protease inhibitors are nearly always needed, while phosphatase inhibitors are required only when investigating phosphorylation states.
Additional preservation methods should be used to prevent your target protein from degradation:
- Work quickly and keep samples cold (consider freezing samples in liquid nitrogen)
- Inhibit or inactivate endogenous proteases and phosphatases
- Add protective or stabilizing compounds, such as reducing agents and enzyme inhibitors
- Stabilize or inactivate proteins by precipitation
Products and resources for protein preservation
Clean up your protein sample
After protein extraction, the protein samples often contain contaminants that are not compatible with protein stability or downstream applications. Dialysis, desalting, and diafiltration (concentration) are three common methods used to remove common contaminants, such as salts and detergents, from protein samples. Depending on the end application requirements, considerations for method choice may include amount of sample input, requirement for functional protein, and processing time. There are a variety of options and formats available for dialysis, desalting, and diafiltration methods.
Dialysis
Dialysis is a classic separation technique that removes small molecules and unwanted compounds from protein in solution by way of selective diffusion through a semi-permeable membrane. A sample and a buffer solution are placed on opposite sides of the membrane. Proteins that are larger than the membrane pores are retained on the sample side of the membrane, but smaller molecules (contaminants) diffuse freely through the membrane until an equilibrium concentration is achieved. Through this technique, the concentration of small contaminants in the sample can be decreased to acceptable levels.
Desalting
Size exclusion chromatography, also described as gel filtration, can be used for removal of salts from samples. In this technique a resin is selected with pores large enough for salts to penetrate but small enough for the protein of interest to enter. This causes contaminants to slow down their rate of migration, and the larger, faster proteins separate from the slower and smaller molecules during gravity flow or centrifugation.
Concentration
Protein concentration is similar to dialysis and uses a semi-permeable membrane to separate proteins from low molecular weight compounds. Unlike dialysis, which relies on passive diffusion, concentration is achieved by forcing solution through membrane by centrifugation. During centrifugation, both buffer and low molecular weight solutes are forced through the membrane where they are collected on the other side (filtrate). Macromolecules (proteins) remain on the sample side of the membrane, where they become concentrated to a smaller volume (retentate), as the reagent is forced across the membrane to the other side.
Products and resources for protein sample clean-up and concentration
Quantify your target protein
Quantifying total protein concentration is an important step in workflows involving isolation, separation, and analysis of proteins by biochemical methods. Assay methods may use fluorescent or colorimetric detection with fluorometers, spectrophotometers, or plate readers. Every protein assay has limitations depending on the application and the specific protein sample analyzed. The most useful features to consider when choosing a protein assay are sensitivity (lower detection limit), compatibility with common substances in samples (e.g., detergents, reducing agents, chaotropic agents, inhibitors, salts, and buffers), standard curve linearity, and protein-to-protein variation.
Colorimetric protein assays
Colorimetric signals can be detected using a microplate reader or spectrophotometer. The most popular colorimetric protein assays are:
- BCA assays: Protein-copper chelation with secondary detection of the reduced copper
- Bradford assays: Protein-dye binding with direct detection of the color change associated with the bound dye
BCA protein assays have an advantage over Bradford assays, as they are compatible with samples that contain up to 5% surfactants (detergents) and are affected much less by protein compositional differences, providing greater protein-to-protein uniformity and accuracy.
Fluorescent protein assays
Fluorescence-based protein quantitation is an alternative to colorimetric methods. Fluorescence detection methods help provide excellent sensitivity, requiring less protein sample, thereby leaving more sample available for your experiment. Fluorescent assays can enable working ranges down to 10 ng/mL compared to enhanced colorimetric assays at 500 ng/mL and 2,000 ng/mL for standard colorimetric protocols. Additionally, read time is not a critical factor, so the assays can be readily adapted for automated high-throughput applications. The fluorescence signal can be detected using a fluorometer or microplate reader.
Products and resources for protein quantitation
Detect and measure your target protein
There are various methods that can be used to detect and measure your target protein depending on your experimental needs. Below are common techniques used to detect and measure proteins from complex mixtures (e.g., lysates, sera) and the typical requirements for each.
| ELISA | Western Blotting | Mass Spec | |
|---|---|---|---|
| Advantages |
|
| |
| Sensitivity | <5–10 pg/mL | Low femtogram to high attogram* | Attomolar range (1018) |
| Lysis buffer compatibility |
|
|
|
| Typical total protein required | 0.1–1 µg/mL | 1–50 µg | <1 µg |
| Equipment required | Plate reader | X-ray film or CCD imaging equipment | Mass spectrometer |
| *With high sensitivity HRP substrates, such as SuperSignal West Atto Ultimate Sensitivity Substrate | |||
Recommended equipment
ELISA measurement and analysis
Multiskan Sky Microplate Spectrophotometer
In addition to reliable ELISA measurements, perform UV-Vis photometric research applications such as DNA, RNA, and protein analysis with the Thermo Scientific Multiskan Sky Microplate Spectrophotometer. The Multiskan Sky reader features a broad wavelength range (200–1,000 nm) path length correction and a fast-reading speed. Its intuitive touchscreen user interface, on-board software, and built-in protocols let you run quick measurements directly from the instrument. Alternatively, with any instrument purchase you can use our unlimited license for our easy-to-use Thermo Scientific SkanIt Software, with access to our extensive online library of ready-made protocols.
Western blot documentation and analysis
iBright Imaging Systems
The iBright 1500 Imaging Systems are powerful and easy-to-use, providing sensitive, streamlined, multimode image capture for gel and western blot documentation. The iBright FL1500 Imaging System is capable of easily capturing 4-plex images. It features a large capacitive touch-screen interface and intelligently designed software.
Proteomics mass spectrometry applications
Orbitrap Astral Zoom Mass Spectrometer
The Thermo Scientific Orbitrap Astral Zoom Mass Spectrometer delivers increased throughput, great proteome coverage, enhanced sensitivity, and precise quantitation for a variety of samples, ranging from single cells to spatial proteomics, immunopeptidomics, and plasma.
Products and resources for protein quantitation
Extract your target protein from the sample
The first step in protein analysis is cellular extraction, which requires liberation of protein from the sample source. Whether using mechanical or detergent-based extraction methods, this process inevitably lyses the cell and contributes to the degradation or destabilization of proteins. Therefore, the quality of the data obtained from protein samples directly depends upon the integrity of the protein during the extraction process. For example, some extraction methods may be efficient at cell lysis and solubilization of cell contents but are protein-denaturing, thereby preventing detection and analysis of native protein interactions.
Sample types
Cultured mammalian cells, mammalian tissues, and primary cells are frequently used as sources of physiologically relevant endogenous proteins (including post-translational modifications) as well as overexpression systems (transient or stable). When extracting protein from mammalian tissues, some gentle means of enzymatic and/or mechanical disruption is required to help separate cells from the more complex tissue matrix. For cultured mammalian cells and primary cells that only have a plasma membrane separating the cell contents from the environment, reagents containing detergents can disrupt the protein-lipid membrane bilayer, making total protein extraction relatively easy. Other organisms that are commonly studied or used for recombinant protein expression systems include bacteria, yeast, and plants. These cell types contain cell walls that require additional enzymatic or mechanical disruption to efficiently release their protein content. However, detergent-based solutions have been developed to effectively extract and solubilize protein from these cells without the need for mechanical disruption, which is especially important when processing higher sample numbers or when automating extraction and purification protocols.
Sub-cellular fractionation
For many studies, generating whole cell lysates is an easy, straightforward way to prepare a soluble protein sample for direct detection or for further purification or fractionation. However, the yield or enrichment of a specific protein can be improved significantly if the cell is fractionated into different compartments or organelles before protein extraction.
Mechanical lysis usually disrupts all cellular compartments, thereby making it difficult to isolate specific cellular fractions. However, by carefully optimizing reagents, stepwise differential detergent procedures have been developed to separate nuclear, cytosolic, and membrane protein fractions. With this strategic approach, hydrophobic membrane proteins can be solubilized and separated from the hydrophilic proteins, and intact nuclei, mitochondria, and other organelles can be isolated for direct study or separate protein extraction.
Pro tip
Analyze a sample of the solubilized protein and the insoluble fractions by SDS-PAGE to determine the efficiency of the protein extraction method used.
Products and resources for protein extraction
Preserve your target protein
Cell lysis disrupts cell membranes and organelles, resulting in proteolytic activity that can reduce protein yield and function. To prevent degradation of extracted proteins and maintain their activity, protease and phosphatase inhibitors are frequently added to lysis reagents.
Protease inhibitors function by binding to protease active sites. Due to the differences in the proteolytic mechanisms, no single compound can effectively inhibit all proteases, and therefore, a mixture or cocktail of several different inhibitor compounds is needed to help ensure that protein extracts do not degrade before downstream analyses. Typical cocktails include small molecule inhibitors of serine, cysteine, and aspartic acid proteases as well as aminopeptidases and metalloproteases. While some inhibitors are irreversible, many are reversible and require their continued presence in the crude sample until further purification removes the threat of proteolytic activity.
Likewise, phosphatases vary so an effective phosphatase cocktail containing inhibitors for serine, threonine, tyrosine, acidic, and alkaline phosphatases is recommended to preserve fragile phosphorylation post-translational modifications.
Most researchers use a mixture of several different inhibitor compounds to help ensure the protein extracts do not degrade before analysis of target interest. Protease inhibitors are nearly always needed, while phosphatase inhibitors are required only when investigating phosphorylation states.
Additional preservation methods should be used to prevent your target protein from degradation:
- Work quickly and keep samples cold (consider freezing samples in liquid nitrogen)
- Inhibit or inactivate endogenous proteases and phosphatases
- Add protective or stabilizing compounds, such as reducing agents and enzyme inhibitors
- Stabilize or inactivate proteins by precipitation
Products and resources for protein preservation
Clean up your protein sample
After protein extraction, the protein samples often contain contaminants that are not compatible with protein stability or downstream applications. Dialysis, desalting, and diafiltration (concentration) are three common methods used to remove common contaminants, such as salts and detergents, from protein samples. Depending on the end application requirements, considerations for method choice may include amount of sample input, requirement for functional protein, and processing time. There are a variety of options and formats available for dialysis, desalting, and diafiltration methods.
Dialysis
Dialysis is a classic separation technique that removes small molecules and unwanted compounds from protein in solution by way of selective diffusion through a semi-permeable membrane. A sample and a buffer solution are placed on opposite sides of the membrane. Proteins that are larger than the membrane pores are retained on the sample side of the membrane, but smaller molecules (contaminants) diffuse freely through the membrane until an equilibrium concentration is achieved. Through this technique, the concentration of small contaminants in the sample can be decreased to acceptable levels.
Desalting
Size exclusion chromatography, also described as gel filtration, can be used for removal of salts from samples. In this technique a resin is selected with pores large enough for salts to penetrate but small enough for the protein of interest to enter. This causes contaminants to slow down their rate of migration, and the larger, faster proteins separate from the slower and smaller molecules during gravity flow or centrifugation.
Concentration
Protein concentration is similar to dialysis and uses a semi-permeable membrane to separate proteins from low molecular weight compounds. Unlike dialysis, which relies on passive diffusion, concentration is achieved by forcing solution through membrane by centrifugation. During centrifugation, both buffer and low molecular weight solutes are forced through the membrane where they are collected on the other side (filtrate). Macromolecules (proteins) remain on the sample side of the membrane, where they become concentrated to a smaller volume (retentate), as the reagent is forced across the membrane to the other side.
Products and resources for protein sample clean-up and concentration
Quantify your target protein
Quantifying total protein concentration is an important step in workflows involving isolation, separation, and analysis of proteins by biochemical methods. Assay methods may use fluorescent or colorimetric detection with fluorometers, spectrophotometers, or plate readers. Every protein assay has limitations depending on the application and the specific protein sample analyzed. The most useful features to consider when choosing a protein assay are sensitivity (lower detection limit), compatibility with common substances in samples (e.g., detergents, reducing agents, chaotropic agents, inhibitors, salts, and buffers), standard curve linearity, and protein-to-protein variation.
Colorimetric protein assays
Colorimetric signals can be detected using a microplate reader or spectrophotometer. The most popular colorimetric protein assays are:
- BCA assays: Protein-copper chelation with secondary detection of the reduced copper
- Bradford assays: Protein-dye binding with direct detection of the color change associated with the bound dye
BCA protein assays have an advantage over Bradford assays, as they are compatible with samples that contain up to 5% surfactants (detergents) and are affected much less by protein compositional differences, providing greater protein-to-protein uniformity and accuracy.
Fluorescent protein assays
Fluorescence-based protein quantitation is an alternative to colorimetric methods. Fluorescence detection methods help provide excellent sensitivity, requiring less protein sample, thereby leaving more sample available for your experiment. Fluorescent assays can enable working ranges down to 10 ng/mL compared to enhanced colorimetric assays at 500 ng/mL and 2,000 ng/mL for standard colorimetric protocols. Additionally, read time is not a critical factor, so the assays can be readily adapted for automated high-throughput applications. The fluorescence signal can be detected using a fluorometer or microplate reader.
Products and resources for protein quantitation
Detect and measure your target protein
There are various methods that can be used to detect and measure your target protein depending on your experimental needs. Below are common techniques used to detect and measure proteins from complex mixtures (e.g., lysates, sera) and the typical requirements for each.
| ELISA | Western Blotting | Mass Spec | |
|---|---|---|---|
| Advantages |
|
| |
| Sensitivity | <5–10 pg/mL | Low femtogram to high attogram* | Attomolar range (1018) |
| Lysis buffer compatibility |
|
|
|
| Typical total protein required | 0.1–1 µg/mL | 1–50 µg | <1 µg |
| Equipment required | Plate reader | X-ray film or CCD imaging equipment | Mass spectrometer |
| *With high sensitivity HRP substrates, such as SuperSignal West Atto Ultimate Sensitivity Substrate | |||
Recommended equipment
ELISA measurement and analysis
Multiskan Sky Microplate Spectrophotometer
In addition to reliable ELISA measurements, perform UV-Vis photometric research applications such as DNA, RNA, and protein analysis with the Thermo Scientific Multiskan Sky Microplate Spectrophotometer. The Multiskan Sky reader features a broad wavelength range (200–1,000 nm) path length correction and a fast-reading speed. Its intuitive touchscreen user interface, on-board software, and built-in protocols let you run quick measurements directly from the instrument. Alternatively, with any instrument purchase you can use our unlimited license for our easy-to-use Thermo Scientific SkanIt Software, with access to our extensive online library of ready-made protocols.
Western blot documentation and analysis
iBright Imaging Systems
The iBright 1500 Imaging Systems are powerful and easy-to-use, providing sensitive, streamlined, multimode image capture for gel and western blot documentation. The iBright FL1500 Imaging System is capable of easily capturing 4-plex images. It features a large capacitive touch-screen interface and intelligently designed software.
Proteomics mass spectrometry applications
Orbitrap Astral Zoom Mass Spectrometer
The Thermo Scientific Orbitrap Astral Zoom Mass Spectrometer delivers increased throughput, great proteome coverage, enhanced sensitivity, and precise quantitation for a variety of samples, ranging from single cells to spatial proteomics, immunopeptidomics, and plasma.
Products and resources for protein quantitation
Protein preparation videos
This webinar highlights different membrane protein solubilization strategies and which enrichment or purification is necessary depending upon the downstream mode of detection or analysis.
Resources
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